Fermented oats are gaining popularity due to their nutritional value and the increasing consumer demand for health-conscious foods. These oats are believed to offer enhanced phytochemical and nutritional profiles compared to unfermented oats. The increased nutritional content of fermented oats is associated with various health benefits, including anti-inflammatory and antioxidant activities, which could potentially reduce the risk of chronic diseases. Further investigations are warranted to elucidate the nutritional benefits of fermented oats in human nutrition. This mini review provides a comprehensive overview of fermented oat products available on the market and the various production methods employed for fermenting oats. Furthermore, this review investigates how fermentation affects the chemical composition and biological functions of oats. Additionally, this manuscript presents some future perspectives on fermented oat products by discussing potential research directions and opportunities for further development. The findings presented in this review contribute to the expanding body of knowledge on fermented oats as a promising functional food, paving the way for future studies and applications in the field of nutrition and health. Fermented foods are products made through microbial action and the enzymatic conversion of food components . During Avena sativa L.) are considered one of the healthiest foods worldwide and are associated with numerous health benefits, including reducing the risk of cardiovascular diseases, type 2 diabetes, gastrointestinal disorders, and cancer .,58.57,58Oats stand out among other cereals due to their elevated lipid content ranging from 2% to 13% . The lipLactobacillus acidophilus yielded substantial quantities of various B vitamins , alongside the presence of vitamin E . This reduction in phytate content during fermentation can significantly enhance the solubility of iron, zinc, and calcium [Phytate, an anti-nutrient component, strongly binds to these minerals, reducing their bioavailability for absorption within the gastrointestinal tract . However calcium . FurtherOats contain both soluble fiber in the form of β-glucan and insoluble fiber, along with other minor components like arabinoxylan and xyloglucan ,68. The Bifidobacterium DSM 20456. However, when fermented with Lb. reuteri ATCC 55730 and Lb. acidophilus DSM 20079 or with mixed cultures, no significant impact on β-glucan content was observed. Furthermore, preliminary studies have indicated that the absence of added sugar during fermentation may lead to a notable decrease in β-glucan content, potentially due to preferential utilization by Lactobacilli [Conversely, Bernat and colleagues  reportedβ-glucosidases, cellulose- or xylan-degrading enzymes, and esterase. These enzymes play a role in releasing bound phenols present in the bran into free forms as shown in The health-promoting compounds in oats include not only β-glucan but also a series of phytochemicals, such as C-type avenanthramides (C-type AVAs), A-type avenanthramides (A-type AVAs), triterpenoid saponins, steroidal saponins, phenolic acids, and flavonoids . Most ofAspergillus oryzae var. effuses, Aspergillus oryzae, and Rhizopus oryzae) for the solid-state fermentation of oats. The TPC increased over time, with the highest TPC observed in oats fermented with Rhizopus oryzae after 72 h. The TPC in these fermented oats was approximately twice as high as in unfermented oats. A similar study by Călinoiu and colleagues [Saccharomyces cerevisiae as a starter culture for the solid-state fermentation of oat bran. The TPC reached its highest level on the fourth day, showing an 83% increase compared to unfermented oat bran.Several studies have investigated the impact of fermentation on the TPC of oats. Cai and colleagues  conductelleagues  used SacMonascus anka alone and in combination with Bacillus subtilis. Fermenting oats with Monascus anka for 12 days resulted in an 18-fold increase in TPC. When oats were fermented with a combination of Monascus anka and Bacillus subtilis, there was an even more significant increase, with TPC multiplying 23 times on the fourth day. These findings suggest that using different or multiple microorganisms may have a more pronounced effect on TPC than using a single microorganism.Another study by Bei and colleagues  investigLb. plantarum, Lb. acidophilus, Lb. casei, Lb. bulgaricus, and Streptococcus thermophilus to compare the effects of fermentation on polyphenols. Except for the sample fermented with Lb. casei, all fermented samples showed a decreasing trend in TPC during the later stages of fermentation, especially after 12 h. After 48 h, only the oat sample fermented with Lb. casei showed a 3.14% increase in TPC. Additionally, within 24 h of fermentation, all five strains’ free polyphenols content (FPC) was higher than that of the control sample, while the bound polyphenol content (BPC) exhibited the opposite trend. This observation can be attributed to the microbial metabolic activity that converts BPC into FPC.However, a study by He and colleagues  reportedp-coumaric, o-coumaric, caffeic, and sinapic acids  in free, conjugated, and bound phenolic fractions of unfermented and fermented oats. Their findings revealed significant increases in specific phenolic acids within the free phenolic fractions of fermented oats. Notably, gallic acid, chlorogenic acid, vanillic acid, and ferulic acid exhibited fold increases of 13.05, 47.31, 18.64, and 28.22, respectively. In the bound phenolic fractions, vanillic acid was undetectable in fermented oats, while p-coumaric acid, sinapic acid, and ferulic acid were the major components, showing fold increases of 44.19, 51.38, and 40.35, respectively. The conjugated phenolic content ranged from 20.60 ± 2.31 to 438.42 ± 5.66 mg/kg, with a nearly 100-fold increase observed in chlorogenic acid content.Several studies have investigated the individual phenolic acids in fermented oats. One study by Soycan and colleagues  examinedA. oryzae var. effuses-fermented oats showed approximately 3- and 9-times higher contents of caffeic acid and ferulic acid, respectively, compared to native oats. The EA subfraction of A. oryzae-fermented oats exhibited 2.7- and 5.5-times higher levels of caffeic acid and ferulic acid, respectively, than native oats. Both chlorogenic acid and p-coumaric acid increased more than two times in the A. oryzae var. effuses and A. oryzae-fermented oats. However, only caffeic acid and p-coumaric acid significantly increased the EA subfraction of A. niger-fermented oats.In another study, Cai and colleagues  examinedS. cerevisiae as a starter culture for a fermentation period of 0 to 6 days. They observed notable increases in specific phenolic acids during fermentation. On day 4, there was a significant increase of 68.81% in p-coumaric acid, followed by a 21.24% increase in ferulic acid and a 34.55% increase in dihydroxybenzoic acids. Sinapic acid exhibited the highest increase (+57.07%) on day two compared to the control.4.4.3. FlavonoidsFurthermore, Călinoiu and colleagues  investigA. oryzae var. effuses, A. oryzae, and A. niger. The concentrations of flavonoids in these fermented oats were 7893.1, 5749.9, and 5285.2 mg of rutin equivalents (RE) per 100 g of dry weight (DW), respectively, compared to the native oat content of 3714.8 mg of RE per 100 g of DW. Notably, luteolin, initially detected only in lesser amounts in native oats, increased after fermentation with all three fungi.Although typically found in cereals in small quantities, flavonoids play an essential role in their health benefits. As seen in Monascus-fermented oats using HPLC. Catechin was initially detected in low amounts only in the free and conjugated fractions, but after fermentation, its content in these two fractions significantly increased. Rutin and quercetin, initially present in low amounts or undetected in raw oats, were found in the conjugated fraction of fermented oats. The concentrations of rutin and quercetin in the conjugated fraction were 355.07 mg/kg and 42.35 mg/kg, respectively. This suggests that fermentation enhances the presence of these flavonoids in oats.In another study by Bei and colleagues , four flLb. plantarum, Lb. acidophilus, Lb. casei, Lb. bulgaricus, and S. thermophilus). Following a 4 h fermentation period, the levels of free flavonoids increased in samples fermented using all four bacteria, except for L. casei, whereas the bound flavonoids decreased. Initially, L. casei displayed an opposite trend but eventually aligned with the same pattern as the other strains during the later stages of fermentation. Notably, at each fermentation time point, the content of free flavonoids in L. casei was significantly higher than that in the other strains, reaching 202.60 mg RE/L at 48 h. This represented a 20.47% increase relative to the unfermented samples.A study by He and colleagues  reportedp-coumaric acid (referred to as 2p or A), ferulic acid (referred to as 2f or B), and caffeic acid (referred to as 2c or C) [Avenanthramides, distinctive phytochemicals found in oats, comprise an amide conjugate formed between anthranilic acid and hydroxycinnamic acids. There are three major types of C-type avenanthramides (AVAs): those derived from 5-hydroxyanthranilic acid with 2c or C) .S. cerevisiae, a significant increase in avenanthramides  was observed on day 4, with respective increases of 48.45%, 42.10%, and 34.50% compared to unfermented oats [A. oryzae var. effuses, A. oryzae, and A. niger), the contents of 2f, 2p, and 2c were also increased [Two studies have investigated the impact of oat fermentation on the content of avenanthramides ,52. In oFermented oats are associated with a wide range of health benefits owing to their improved nutrient bioavailability and increased levels of beneficial compounds. These advantages encompass enhanced blood sugar regulation, assistance in weight management, lowered cholesterol levels contributing to improved cardiovascular health, a decreased risk of specific types of cancer, and therapeutic effects in celiac disease treatment .Lb. acidophilus, S. thermophilus, and Bifidobacterium bifidium. Chemical composition analysis and the evaluation of various biomarkers were conducted to assess the impact of fermented oat milk. These biomarkers included the liver-to-body weight ratio, pancreas-to-body weight ratio, glucose levels, triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC), as well as the activity of liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The findings demonstrated diverse effects across the evaluated biomarkers. The administration of FOM led to reduced glucose levels (28.3% reduction), improved lipid profiles characterized by lower TC (reduced from 155.33 ± 1.37 to 115.33 ± 0.71 mg/dL), TG (reduced from 89.33 ± 0.14 to 50.00 ± 0.31 mg/dL), and LDL-C (reduced from 76.00 ± 1.22 to 66.33 ± 0.54 mg/dL), and higher HDL-C levels (increased from 27.00 ± 0.71 to 45.33 ± 0.98 mg/dL). Additionally, it resulted in decreased liver enzyme activity and alterations in the composition of the intestinal microbiota, characterized by increased counts of Lactobacilli and Bifidobacteria. The study sheds light on the potential advantages associated with consuming fermented oat milk, particularly in ameliorating glucose control, enhancing lipid metabolism, promoting liver health, and influencing the composition of the intestinal microbiota, in rats with diabetes (Gohari and colleagues  conductediabetes .Lactobacillus plantarum-fermented oats and fermented oats supplemented with Sidr honey on type 2 diabetes in rats. Diabetes was induced by administering a solution of streptozotocin (STZ) in 0.1 M citrate buffer with a pH of 4.5 at a dosage of 45 mg kg−1 BW. After 72 h of fermentation, the honey-supplemented fermented oats showed more desirable effects on TPC (increased from 1.41 to 1.64 mg GAE g−1). The antioxidant capacity of the fermented oats was significantly increased, as indicated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) measurements  of DPPH and from 7.68 to 11.73 µmol of TE g−1 of ABTS). Oral administration of the fermented oat extract for six weeks effectively improved diabetic complications in rats, including blood glucose levels, lipidemia, liver and kidney functions, and oxidative stress. For instance, the administration of fermented oats demonstrated beneficial effects on blood glucose levels during the 0th, 3rd, and 6th weeks of treatment, with values of 308.5 ± 22.13 mg dL−1, 244.83 ± 28.82 mg dL−1, and 232.50 ± 15.24 mg dL−1, respectively. When fermented oats were supplemented with honey, the effects were even more pronounced, resulting in blood glucose levels of 320.83 ± 39 mg dL−1 for the 0th week, 225.17 ± 18.35 mg dL−1 for the 3rd week, and 211.50 ± 32.66 mg dL−1 for the 6th week The study demonstrated that the fermented oat extract, especially when supplemented with Sidr honey, exhibited significant antioxidative and ameliorative effects against type 2 diabetes. The presence of bioactive compounds such as TPC and GABA in the fermented oats and the synergistic effects of Sidr honey likely contributed to the observed benefits.In another study, Alharbi and colleagues  investigLactobacillus plantarum for 72 h. Diabetes was induced in the rats via the intravenous administration of STZ dissolved in 0.1 M citrate buffer at a pH of 4.5. The dosage of STZ used for induction was 45 mg kg−1 BW. The fermented oat extract, LFOE, was administered orally to diabetic rats for six weeks. The results showed that fermented and unfermented oat extracts effectively reduced blood glucose levels. LFOE had slightly better effects, but both extracts significantly improved insulin sensitivity and reduced lipid levels. The fermentation time was crucial, as longer fermentation increased the viable Lb. plantarum count and the production of beneficial compounds like GABA and phenolic compounds. The fermentation process decreased β-glucan content but did not significantly affect the effectiveness of LFOE in improving hyperglycemia. Both extracts showed hepatoprotective and nephroprotective effects, improving liver enzymes and serum proteins. The fermentation process increased the extracts’ TPC and antioxidant capacity.Furthermore, Algonaiman and colleagues  investigOverall, the existing studies suggest that fermented oats have significant antidiabetic, hypolipidemic, hepatoprotective, and antioxidant effects, which could be attributed to the production of bioactive compounds during fermentation. Further research is needed to identify the active components in fermented oats and understand the underlying mechanisms of these effects.p-coumaric, caffeic, and ferulic acids as the primary compounds responsible for lipase inhibition (p-coumaric and ferulic acids displaying more potent inhibitory effects than caffeic acid. Synergistic interactions were observed at high concentrations (250 μg/mL) but additive effects occurred at low concentrations (50 μg/mL), suggesting that lower concentrations of these phenolic acids contribute additively to lipase inhibition. The study proposed fermented oats and polyphenol-rich foods as potential complementary therapies for obesity treatment. However, further research, particularly human studies, is needed to validate these findings in human subjects.Studies have shown that pancreatic lipase plays a key role in splitting triglycerides into bioavailable glycerol and fatty acids in the gastrointestinal tract in humans . Reducinhibition . These pA positive association exists between elevated serum cholesterol levels and increased susceptibility to ischemic heart disease . NumerouPediococcus damnosus 2.6, resulted in a fermented product with a “ropy” characteristic (thick stringy texture). Human participants with moderately elevated cholesterol levels were divided into three groups: one consuming the fermented dairy product and the other two groups consuming different oat-based products. Fecal bacteria count and Bifidobacterium ssp. were measured to assess the prebiotic potential of the products. The group consuming the ropy oat-based product with a daily intake of 3.5 g of β-glucans showed a significant decrease in total cholesterol levels compared to the dairy-based product group. However, the other oat-based product with a daily intake of 3.0 g of β-glucans did not significantly affect plasma lipids. The concentration of β-glucans in the products influenced their effectiveness, and the presence of microbial β-glucans in the ropy oat-based product potentially contributed to lipid reduction. The study suggested that consuming approximately 3.5 g of native glucans daily through a fermented, ropy, oat-based product can effectively lower plasma lipid levels. Future research should investigate the physiological impact of isolated microbial β-glucans on plasma lipid reduction, and the fermented oat-based product shows potential as a prebiotic based on increased fecal Bifidobacterium ssp. counts.Mårtensson and colleagues  conducteRhizopus oryzae 3.2751, which exhibited high polyphenol content. The EA subfractions demonstrated significant anti-cancer activity against four human cancer cell lines  while showing minimal toxicity toward normal cells.Fermented oats, which have improved nutritional properties and are rich in polyphenols, were investigated by Zhang and colleagues  for theiMale and female KM mice (5–6 weeks old) and female BALB/c nude mice (5–6 weeks old) were used in the animal model. The mice were intraperitoneally administered EA subfractions dissolved in corn oil at graded doses of 5, 10, 20, 40, and 60 mg/kg body weight daily for 24 days, with an injection volume of 10 mL/kg body weight. The control group received intraperitoneal injections of corn oil alone. In vivo experiments conducted on tumor-bearing mice revealed the inhibitory effects of the extracts on tumor growth without causing significant toxicity. The study further investigated the impact of the extracts on HepG2 cells (human hepatocellular carcinoma cells) to elucidate the underlying mechanisms. The extracts induced cell cycle arrest at the G2/M phase, triggering apoptosis in HepG2 cells. They also led to a reduction in mitochondrial membrane potential and modulated the expression of apoptosis-related proteins. Moreover, the extracts increased intracellular reactive oxygen species (ROS) levels and activated the ROS/JNK signaling pathway, contributing to the induction of apoptosis. These findings suggest that the anti-cancer effects of the fermented oat extracts involve multiple mechanisms, including cell cycle arrest, apoptosis, and the regulation of ROS signaling pathways .Celiac disease (CD) is an immune-mediated condition triggered by gluten exposure in individuals with specific genetic predispositions . Oats haL. plantarum WCFS1 and its impact on nutritional serum biomarkers and intestinal microbiota composition in adults with CD. The study involved ten celiac adults (aged 22–64 years) adhering to a gluten-free diet for at least two years. They were randomly divided into two groups: the treated group received 200 mL/day of SOFB, and the control group received a gluten-free placebo. The intervention period was six months, during which participants maintained their gluten-free diet. Clinical and immunological markers were monitored at 0, 2, 4, and 6 months. SOFB consumption was well-tolerated and safe, as it did not induce mucosal damage or immunoglobulin A antibodies against tissue transglutaminase (IgA anti-tTG antibody) production. In terms of its effects, SOFB did not lead to weight changes, but it positively impacted cholesterol levels, increasing beneficial gut bacteria like lactobacilli and Ruminococcus while decreasing inflammatory markers. The results suggest that SOFB could be a promising adjunct in celiac disease treatment, but further research in a larger population is needed.In an intervention study, Aparicio-García and colleagues  investigThe market currently has only a few fermented oat products available, with European countries  and the United States leading in their promotion and production. Bacteria have been more commonly used in developing fermented oats compared to fungi and yeasts. However, research on fermented oats and their health benefits is still in its early stages, mostly relying on limited in vitro, rodent, and human studies. To better understand the health effects of fermented oats, more research is needed, especially in human in vivo models.Previous studies have shown that fermentation can increase the levels of certain C-type AVAs and phenolic acids in fermented oats compared to unfermented ones. However, the impact of fermentation on other bioactive compounds like steroidal saponins ,106, triWhile some studies have indicated beneficial effects of fermented oats on conditions such as diabetes, hyperlipidemia, obesity, cancer, and celiac disease, based on in vitro and in vivo experiments, as well as human studies using extracts and fermented oat beverages, further research using whole fermented oats is essential to establish their potential as therapeutic interventions for these health conditions. These comprehensive investigations will help us better understand the therapeutic benefits of fermented oats and their potential role in managing these pathological conditions. Additionally, exploring the individual contributions of probiotics, β-glucan, and phytochemicals in fermented oats, as well as their potential additive or synergistic effects, is essential to maximize their positive impact on human health. Comprehensive investigations are thus warranted to unlock the full potential of fermented oats as a functional and health-promoting food.


PIC results showed that eight loci were all highly polymorphic. The means of Ho and He of pop2 were the highest, which were 0.646 and 0.717, respectively. The PCoA analysis demonstrated that the samples from three conservation farms were mixed together. The phylogenetic tree showed that pop 2 and pop 3 were closely related. The phylogenetic tree results clustered that 272 donkeys were divided into six groups. AMOVA analysis showed that the genetic variation was mainly concentrated within the population and the genetic differentiation among the populations was low. Fst values between populations also indicated that genetic differentiation between populations was too small to be considered. This indicated a low probability of inbreeding within the population. And this also showed that the conservation and breeding of Dezhou donkeys in recent years have achieved excellent results. The investigation of genetic diversity in three Dezhou donkey original breeding farms can provide reference data for the selection and breeding of excellent breeds of Dezhou donkey.Dezhou donkey is one of the excellent large donkey breeds in China. In our study, eight microsatellite markers were used to genotype from each of three populations of Chinese Dezhou donkeys: 67 individuals in Liaocheng (pop1), 103 individuals in Binzhou 1 (pop2), and 102 individuals in Binzhou 2 (pop3), in order to study the genetic diversity of these populations. A total of 213 alleles were detected, and the  In 2011, Dezhou donkeys were included in the national livestock and poultry genetic resources protection list. Dezhou donkey is an excellent local livestock breed in Shandong Province, China. After careful cultivation, it has gradually formed a unique local breed. With the development of agricultural mechanization, donkeys had gradually lost their traditional role as working animals3 and become dual-purpose farm animals1. Statistics from China’s Statistical Yearbook showed that the number of donkeys dropped from 9.444 million in 1994 to 2.324 million in 2020. Dezhou donkey breed was facing the problems of rapid population decline and germplasm quality degradation. Therefore, it is urgent to preserve the genetic diversity and variability of Dezhou donkey.Dezhou donkey Fig.  is one o4, and according to Shandong Province local standard ‘Technical Specification for Dezhou Donkey Frozen Semen Producing’, a tube of frozen semen for donkeys is 0.5 mL. Therefore, with the application of artificial insemination and other breeding techniques in donkey farms, this resulted in the slaughter of most of the male donkeys at the original breeding farms. Simultaneously, the purchased semen by the breeding farm may not be purebred Dezhou donkeys, which can easily result in crossbreeding with other breeds. This has led to the decline of Dezhou donkey germplasm resources. Therefore, it is very important to investigate the genetic diversity in the Dezhou donkey original breeding farm. At present, the genetic diversity among different Dezhou donkey populations has not been analyzed and reported, this still needs further study.Livestock breeding industry is the foundation of animal husbandry development and the important embodiment of the core competitiveness. Donkeys produce an average of 30 to 90 mL of semen per ejaculation5. Microsatellite markers have been widely applied to analyze genetic diversity and variability of livestock. In the study of equine species, Zeng et al.6 used microsatellite markers to analyze the genetic diversity and structure of Guanzhong horses. Sun et al.7 assessed the genetic diversity of donkeys and horses using microsatellite markers. Kefena et al.8, Zhang et al.9, and Aranguren-méndez et al.10 have analyzed the genetic diversity and variability of different donkey breeds. However, the analysis of genetic diversity among different Dezhou donkey populations by microsatellite markers has not yet been done and reported.Microsatellite DNA is widely distributed in chromosomes, which has the advantages of high polymorphism, conservatism, and easy detectionIn this study, the polymorphism of eight microsatellite loci in Dezhou donkey was investigated by capillary electrophoresis sequencing, and genetic diversity as well as genetic distance in three Dezhou donkey populations were analyzed. This provides the basis for the conservation of biodiversity and genetic resources of the Dezhou donkey.The experimental animals and methods used in this study were approved by the Animal Policy and Welfare Committee of Liaocheng University (no. LC2019-1). The care and use of laboratory animals were in full compliance with local animal welfare laws, guidelines and policies.11.Samples were collected from three Dezhou donkey original breeding farms. One of the three breeding farms is located in Liaocheng City, Shandong Province, and two are located in Binzhou City, Shandong Province. A total of 272 individuals were randomly collected from three breeding farms, including 67 individuals in Liaocheng (pop1), 103 individuals in Binzhou 1 (pop2), and 102 individuals in Binzhou 2 (pop3), which represent the major genetic resources of Dezhou donkey. The farm location is shown in Fig. 12.DNA was extracted from blood using the M5 FlexGen Blood DNA kit . The DNA purity (OD260/OD280) was measured by spectrophotometer , and the quantity of genomic DNA extracted was measured using 1% agarose gel13, eight microsatellite loci were analyzed. The primer sequences are shown in Table 2O, 1 µL forward primer , 1 µL reverse primer  and 2 µL genomic DNA11. PCR reaction procedure: 35 cycles of predenaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing for 50 s, and extension at 72 °C for 30 s, it was extended at 72 °C for 40 min and stored at 4 °C14.According to the results of previous studies12, the reagents were as follows: Applied Biosystems POP-7 Polymer and Hi-Di Formamide, running parameters: Run Voltage: 15,000, Injection Voltage: 1600, Injection Duration: 15, Temperature: 63, Laser Power: 12. Fluorescently labeled fragments were detected and sized using GeneMarker version 2.6.4 .PCR products were detected on 1% agarose gels, and those with specificity were separated by capillary electrophoresis using an ABI 3730xlDNAAnalyzer Ho), Expected Heterozygosity (He), No. Effective Alleles (Ne), No. Alleles (Na), number of private alleles (NPA) and principal coordinates analysis (PCoA) were calculated using GenAlEx version 6.5115. Polymorphic information content (PIC), the number of alleles (K), and the number of individuals at the locus (N) were calculated using Cervus version 3.0.716. Percentage of estimated genetic distance from the Fst Values for Dezhou donkeys from the three populations were calculated using GenAlEx version 6.5115. Arlequin version 3.1 software was used for AMOVA analysis17. Popgene version 1.32 software was used to generate the phylogenetic tree of three Dezhou donkey populations12. Mega version 11.0 software and PowerMarker version 3.25 software were used to generate the phylogenetic tree of 272 Dezhou donkeys19.The Hardy–Weinberg equilibrium (HWE), Observed Heterozygosity . This study in accordance with the arrival guidelines.20 constructed a set of multiplex PCR systems for horses, which can amplify multiple microsatellite marker loci by a single PCR reaction, with the advantage of saving time and effort, but the study has not been reported by relevant studies on donkeys, and the method needs our further study on donkeys.Eight microsatellite markers were amplified in three Dezhou donkey populations. Figure PIC is an indicator of gene abundance and its level reflects the diversity of the genetic base of a breed, the PIC reflects the genetic variation of microsatellite loci21. All eight loci in population 2 were highly polymorphic, all loci except TKY337 and HMS6 were highly polymorphic in pop 1, and all loci except HMS7 were highly polymorphic in pop 3 (PIC > 0.5), indicating that they were all highly polymorphic. In this study, abundant polymorphism has been observed for all three populations. Zhang et al.9 found that the mean PIC for the seven polymorphic loci of the Dezhou donkey population was 0.4561, in the present study the PIC results for all three of our populations were higher than that result, this may be due to the large sample size. Consistent with the results of Kim et al.7, AHT4, ASB23, HMS2 and HMS3 were highly polymorphic in the donkey population.10. found that only one private allele (HTG6) had a frequency greater than 5% in Encartaciones breed. This may be due to the large number of populations selected for this study and the relatively long geographical distance of the populations.Except for HMS7 and HMS6, the remaining loci showed different levels of Hardy–Weinberg equilibrium in the three populations. Four loci  in population 1 were not in Hardy–Weinberg equilibrium, which may be due to the smaller sample size of pop 1 compared with pop 2 and pop 3. All eight loci in pop 3 were in Hardy–Weinberg equilibrium. Pop 2 had the highest number of private alleles, and the TKY343 locus had eight private allele numbers  to 10.625 (pop 2). The Ne was the highest in the pop 2 (3.787) and lowest in the pop 1(3.366), with a mean of 3.527. The Ho and He of pop 2 were the highest, which were 0.646 and 0.717, respectively. The Ho and He of pop 1 were the lowest, which were 0.498 and 0.646, respectively. Interestingly, these findings were also found in other studies, for example, Aranguren-Méndez et al.10 reported an average He in 13 microsatellite markers ranging between 0.637 and 0.684 for five endangered Spanish donkey breeds, which is basically consistent with our results. Bordonaro et al.22 found that the He of the endangered Pantesco and two other Sicilian donkey breeds was lower than that of our study population, ranging from 0.471 for Pantesco to 0.589 for Grigio.Genetic variability parameters were calculated for the three populations Table . The mea8 explored the population structure of native Ethiopian donkeys (Equus asinus) with equine using the PCoA method, coordinate 1 (24.19%) mainly distinguished Abyssinian, Afar and Haraji donkeys from Ogaden, Omo and Sinnar donkeys, coordinate 2 (19.29%) did not separate Ethiopian donkeys into any group, this is consistent with our results.The PCoA method was performed to investigate possible genetic relationships among Dezhou donkey populations Fig. . The PCo7 found that thoroughbreds overlap with Jeju Halla horses in the phylogenetic tree.Figure 10 found that the genetic distance of the Spanish donkey breeds ranged from 0.057–0.093, this is lower than our results.The genetic distances according to Nei (1972) are summarized in Table 23. AMOVA analysis showed that 0.14382 of the genetic variation occurred among populations and 2.70564 of the genetic variation occurred within the population, indicating that the genetic variation was mainly concentrated within the population and the genetic differentiation among the populations was low (Table The AMOVA analysis is a satisfactory grouping criterion for evaluating the variation within and among populationsIn conclusion, we evaluated genetic diversity in three Dezhou donkey breeding farms using microsatellite markers. The results showed that abundant polymorphism and low inbreeding levels were observed in all three populations, this provides a direction for the conservation of genetic diversity of Dezhou donkeys. However, this study was limited by the low sample size, and further investigation is needed to expand the sample size in the future.Supplementary Tables.


The retrieval of autobiographical memories is influenced by several factors, such as sensory modality and the emotional salience of memory cues. This study aimed at investigating the interaction between sensory modalities  and emotional dimensions  of imagery cues, on the frequency, quality, and age distribution of the autobiographical memories (AMs) elicited.A total of 296 females (aged between 18 and 35 years) received one out of eight brief instructions for olfactory or visual imagery. The participants were asked to create a mental image with either high arousal/positive valence, high arousal/negative valence, low arousal/positive valence, or low arousal/negative valence ; no specific stimulus was mentioned in the instruction.The approach used elicited imagery with autobiographical content in the majority of participants (78%). In terms of frequency, odor imagery, compared to visual imagery, turned out to be more effective at retrieving either unpleasant memories associated with experiences in adulthood, or pleasant childhood memories. In terms of quality, the imagery was rated as less vivid in the olfactory compared to the visual condition . Visual imagery was associated with the experience of more diverse emotions  than odor imagery, which was related primarily with disgust and happiness.Our findings indicate that nonspecific imagery induction is a useful approach in accessing AM.This approach presents promising clinical applications, such as in working with autobiographical memory narratives in psychotherapy.The online version contains supplementary material available at 10.1007/s00426-022-01701-y. Random odors can evoke autobiographical memories ; this is otherwise known as the Proust phenomenon. For instance, a certain odor may transport one suddenly back into the arms of one’s grandmother, who had used a perfume with a similar scent, or alternatively, it might remind another person of an unpleasant experience at the dentist’s.The phenomenon of odors as retrieval cues for AMs has been the subject of various studies  in terms of the emotional dimensions valence and arousal  to investigate the potential of odor imagery as a retrieval cue for AMs and b) to elucidate interactions between emotional dimensions  and sensory modalities of memory cues, on the frequency, vividness, and age distribution , of the AMs reported. We hypothesized that the novel imagery induction approach would facilitate the retrieval of odor-related AMs, compared to classical deliberate cueing methods. Therefore, we assumed that the percentage of participants who would retrieve an AM would be higher than in the previous literature on AM retrieval after presentation of olfactory imagery  with a probability of 1 – β = 0.95, α = 0.05 for the χ2-tests to analyze AM frequency and a minimum sample size of N = 206 would be necessary to detect an effect size of part.η2 = 0.06 (i.e. medium effect) with a probability of 1 – β = 0.95, α = 0.05 for the main effects and interaction effects of sensory modality, valence and arousal of the imagery induction (2 × 2 × 2 ANOVAs) on AM vividness. Since previous literature suggests that not all participants would be able to generate mental images as well as AMs  to account for possible dropouts.The participation requirements  were communicated prior to testing. Twentyeight male participants and nine participants aged > 35 filled out the survey anyway. They were excluded after testing. The participants were recruited via announcements at the university campus and through social media. 97% of the participants had a high school diploma, 86% were university students. Participants reported no mental health problems as indicated by the scores of the brief symptom inventory . Additionally, they indicated whether or not the image referred to an event personally experienced in the past ; e.g., “The imagery reminded me of the smell when I once had to puke after a roller coaster ride”). We analyzed the percentage of participants who reported an AM image. If an AM image occurred, the participants reported their age, when the event had occurred. The events were either classified as childhood memories (age: 0–10 years), memories attributed to adolescence (age: 11–17 years), or more recent memories of adulthood (age: 18–35 years). Finally, the vividness of the imagery experience was rated on a seven-point Likert scale . Reported arousal and valence for AM images were rated as a manipulation check .The groups did not differ concerning the number of participants and mean BSI score . After obtaining written informed consent, the participants were asked for demographic data . Then participants listened to the imagery instruction and named/rated the evoked AM images. Furthermore, participants filled out the BSI;  were examined via χ2-tests. .We compared the frequency of AM images  between the factors Sensory Modality , Valence (pleasant vs. unpleasant), and Arousal  of imagery induction via The effect of imagery induction , Valence (pleasant vs. unpleasant), and Arousal  on the reported vividness of AM imagery was tested using a 2 × 2 × 2 between subjects analysis of variance (ANOVA).χ2 tests.As exploratory analyses, the content of the reported AMs was classified according to five basic emotions: happiness , sadness , anger , anxiety (e.g. accident), and disgust . The emotional content of the memories was classified by two independent psychologists with sufficient interrater agreement (Cohen’s Kappa = 0.61-0.81). The frequency of basic emotion-related content of AMs was compared between the conditions via Finally, as a manipulation check, the effects for imagery induction , Valence (pleasant vs unpleasant), and Arousal  were tested on reported valence and arousal of AM imagery with a 2 × 2 × 2 ANOVA.χ2 (1) = 10.22, p = 0.001, V = 0.186). Also in the pleasant imagery condition more participants reported an AM (85%) compared to the unpleasant imagery condition  = 9.25, p = 0.002, V = 0.177). Regarding interactions between sensory modality, valence, and arousal, the results of the χ2-analyses were in line with our hypothesis and showed that in the unpleasant conditions , more AM images were elicited via olfactory instructions (85%) compared to visual instructions  = 6.39, p = 0.011, V = 0.284, χ2Low (1) = 9.70, p = 0.002, V = 0.362). Furthermore, via visual imagery less AM images were evoked in the unpleasant (56%) compared to the pleasant imagery conditions  = 9.91, p = 0.002, V = 0.361; χ2Low (1) = 5.15, p = 0.023, V = 0.271). All other comparisons were not statistically significant  reported an imagery experience with autobiographical content. Surprisingly, even more AM images were reported in the olfactory imagery condition (85%) compared to the visual imagery condition  = 14.15; p < 0.001; V = 0.307), whereas this was not the case in the visual conditions (χ2(1) = 0.003; p = 0.957; V = 0.004; VP = 9.72% vs VUP = 9.46%). In the odor conditions, childhood-related AMs were almost exclusively reported for pleasant imagery cues (20% of participants reported childhood-related AMs), whereas in the unpleasant conditions almost none of the reported AMs were childhood-related (0.01%). In the unpleasant condition more AMs related to childhood were reported in the visual (9%) compared to the odor imagery condition  = 5.18; p = 0.023; V = 0.184); whereas in the pleasant condition the percentage of childhood-related AMs did not significantly  differ between visual and odor imagery condition (χ2(1) = 2.85; p = 0.091; V = 0.141). See Fig. Regarding the frequency of childhood-related AMs, the conducted ction  = 5.68; p = 0.017; V = 0.199). All other comparisons were not significant . See Fig. In contrast to childhood-related AMs, the results on adolescence-related AMs and adulthood-related AMs are in line with our hypothesis. In the pleasant conditions, a higher percentage of participants reported AMs related to adolescence in the visual (25%) compared to the odor imagery conditions  = 8.10; p = 0.004; V = 0.230), whereas the percentage of participants who reported AMs related to adulthood did not differ between visual (53%) and odor imagery in the pleasant condition  = 0.17; p = 0.677; V = 0.035). Furthermore, the percentage of participants who reported AMs related to adulthood was lower in the visual unpleasant (37%) compared to the visual pleasant imagery condition  = 3.92; p = 0.048; V = 0.164), whereas the percentage of adulthood-related AMs in the odor condition did not significantly differ between pleasant and unpleasant imagery induction (χ2(1) = 1.57; p = 0.210; 0.102). See Fig. In the unpleasant imagery conditions, more adulthood-related AMs were reported in the odor (60%) compared to the visual imagery condition .M = 4.18, SD = 1.35) than in the olfactory imagery conditions . All other main and interaction effects were not statistically significant; p > 0.173). For statistics, see Table The ANOVA revealed a significant main effect for Sensory Modality  than AMs retrieved in the unpleasant conditions . All other effects were not significant .As expected, the ANOVA identified a significant main effect for valence of imagery instruction on the valence ratings for the AM imagery (supplementary Table 2). AMs retrieved in the pleasant imagery conditions were rated as more pleasant  = 8.67, p = 0.004, partη2 = 0.038). All other effects were nonsignificant (p > 0.345). For means and standard deviations see supplementary Fig. 1b and c; for statistics see supplementary Table 2.However, the computed ANOVA for arousal ratings was only partly in line with our instructions. The results revealed significant interactions between Sensory Modality, Valence, and Arousal of imagery instruction (see supplementary Table 2). In the visual imagery condition with unpleasant/high arousal adjectives, AMs were rated more arousing than in the visual imagery condition with unpleasant/low arousal adjectives; This study investigated the effects of sensory modalities  and emotional dimensions  of imagery cues on the frequency, vividness, and age distribution of reported AMs. As a new approach, we used imagery instructions as retrieval cues and not specific odors or pictures  were able to generate a mental image with autobiographical content. In contrast, Willander & Larsson , who useIn terms of the reported age distribution of AMs, most memories were attributed to adulthood/adolescence, whereas only a small percentage of participants (approximately 10%) retrieved childhood-related memories. This is in line with previous research on the reminiscence bump [i.e., the tendency to access more personal memories from young adulthood than from other periods of life . Our new approach of nonspecific induction of olfactory imagery to access AM might be an option as one component of exposure therapy, which so far relied on the administration of real odors . Thus, our results cannot be generalized to other groups. Future studies on sex and age differences in the imagery task are strongly recommended. Further shortcomings of the present investigation concern the imagery instruction. The valence instructions for the imagery worked well; AMs retrieved after pleasant retrieval cues were rated as more pleasant than AMs retrieved after unpleasant retrieval cues. However, descriptions of stimuli inducing high vs. low arousal did not lead to AMs that differed in experienced arousal. Similar findings regarding the arousal dimension have been reported by Schulkind and Woldorf , SheldonTo the best of our knowledge, this is the first study to investigate the effects of sensory modalities and emotional dimensions of imagery as retrieval cues on AMs. Future studies need to replicate the present findings and broaden the research scope. For example, it would be interesting to investigate odor imagery as a retrieval cue in clinical samples with diagnoses of depression, or PTSD.Supplementary file1 (PDF 127 KB)Below is the link to the electronic supplementary material.


With the advent of synthetic biology, scientists are increasingly relying on digital sequence information, instead of physical genetic resources. This article examines the potential impact of this shift on the access and benefit-sharing (ABS) regime of the Convention on Biological Diversity (CBD) and the Nagoya Protocol. These treaties require benefit-sharing with the owners of genetic resources. However, whether “genetic resources” include digital sequence information is unsettled. The CBD conceives genetic resources as genetic material containing functional units of heredity. “Material” implies tangibility, and for some scholars, “functional units of heredity,” undefined in both treaties, mean full-coding sequences. This article argues that digital sequence information obtained from physical genetic resources, full-coding or not, should be treated as genetic resources. Literal construction of the CBD risks eroding its usefulness and the ABS regime. This is because through bioinformatics, sequence information can easily be obtained from genetic resources for utilization, without physically moving them or concluding ABS agreement with owners. The CBD must evolve with scientific progress also because sequence functionality depends on the state of knowledge. These arguments are vindicated by domestic ABS laws equating genetic information with genetic resources; Nagoya Protocol provisions deeming research exploiting the genetic composition of genetic resources as utilization of genetic resources; and CBD provisions requiring the sharing of benefits from the utilization of genetic resources. Moreover, treaty interpretation and case law demand that generic, scientific terms, such as “genetic resources” and “functional units of heredity” be interpreted in an evolutionary manner to capture scientific developments. Synthetic biology, which is a novel technology for the de novo design and creation of biological parts, devices, and systems, as well as the modification of regular organisms , p. 6), , 6], pp.However, a prominent question continues to be whether genetic resources and the ABS regime of the CBD cover digital sequence information, given its intangibility. Under the CBD, genetic resources mean genetic “material” containing “functional units of heredity.” Here, “material” suggests tangibility, and although the CBD and the Nagoya Protocol do not define functional units of heredity, scholars relying on CBD’s negotiating history view them as gene-coding sequences . There is presently a dearth of scientific and legal analyses of the use of digital sequence information in research and development and the implications this may have for ABS obligations established under the CBD and the Nagoya Protocol . Plasmodium vivax .Aware of the above challenges, the Conference of the Parties to the CBD (COP) in Decision CBD XIII/16 taken in Cancun, Mexico, at its thirteenth meeting in December 2016 , Acting on its mandate under COP Decision XIII/16 of 2016, AHTEG convened in February 2018 to discuss the nexus between digital sequence information and terms used in the CBD and the Nagoya Protocol . As part of that process, parties and other stakeholders were invited to submit views on whether and how domestic ABS systems address digital sequence information on genetic resources, including the sharing of benefits from their commercial and non-commercial uses.15In early 2020, the required CBD-commissioned studies were published namely: “Digital sequence information on genetic resources: concept, scope and current use,” (Study 1) ; inter alia, scientific and technical matters concerning the global biodiversity framework and synthetic biology; Part 1 of the third meeting of WG2020  to negotiate the first draft of the global biodiversity framework and consider feedback from AHTEG on how to reconcile the disparate positions on the sharing of benefits from the utilization of digital sequence information on genetic resources; and Part 1 of the fifteenth meeting of COP , with some in-person participation, leading to adoption of the Kunming Declaration  to address, In-person meetings, that is, Part 2 of SBSTTA-24 and Part 2 of WG2020-3, among others, resumed from 14 to 29 March 2022 in Geneva, Switzerland, to prepare the groundwork for the adoption of the post-2020 global biodiversity framework during Part 2 of COP-15 scheduled for Montreal, Canada, in December 2022. Numerous recommendations and decisions were made for consideration by COP-15. There was consensus on the way forward and intersessional work on digital sequence information, as well as on indicators and targets for the global biodiversity framework, with agreement to hold a fourth meeting of WG2020 (WG2020-4) in June 2022 in Nairobi, Kenya .Going back to COP-14, there were conflicting views on the applicability of the ABS regime to digital sequence information. The African Union, the African Group, the Group of Like-Minded Megadiverse Countries, and the International Indigenous Forum on Biodiversity all insisted that such information is within the ambit of the international ABS regime . By contDeliberations at AHTEG’s meeting, which informed the SBSTTA’s recommendation, exhibited similar polarity of views that highlights the continuing controversy over the applicability of the CBD and the Nagoya Protocol to digital sequence information. The CBD-commissioned studies on digital sequence information further reiterate the problem posed by digital sequence information for CBD objectives, particularly benefit-sharing, calling for more studies to explore the topic. Without rules or clarity in rules for the sharing of benefits from the use of digital sequence information on genetic resources, provider states may restrict access to such resources, even for non-commercial research that supports biodiversity conservation. Also, as some statesThe present article contributes to efforts aimed at resolving the issue raised by digital sequence information for the CBD and enhancing clarity for the adoption or modification of domestic ABS regimes. The article advances the central thesis that digital sequence information obtained from regular genetic resources should be treated in like manner as physical equivalents and, therefore, considered as genetic resources under the CBD. A rigid construction of the CBD excluding digital sequence information, full-coding or otherwise, from the meaning of genetic resources due to intangibility, would undermine the ABS obligation of the CBD. Although the ultimate aim of synthetic biologists is to design their own sequences ,To buttress the above arguments, this article relies on domestic ABS laws, as well as expert reports that consider digital sequence information as genetic resources; Nagoya Protocol provisions that deem research and development exploiting the “genetic and/or biochemical composition of genetic resources” as the utilization of genetic resources; and CBD provisions requiring equitable sharing of benefits arising from the utilization of genetic resources. Significantly, reference is made to treaty interpretation and case law, including judicial, as well as quasi-judicial decisions. These latter sources require that vague, generic, and technical terms, such as “genetic resources” and “functional units of heredity” be given evolutionary interpretations in light of unfolding scientific developments.Following this introduction, the article examines the meaning, nature, and importance of genetic sequence information. Next, it analyzes the objectives and conceptual premises of the CBD and the Nagoya Protocol. Also examined here are notions of biological and genetic resources, functional units of heredity, actual or potential value, utilization of genetic resources, and access to genetic resources. Building on these, as well as CBD deliberations, recent commissioned studies and peer-reviewed articles on digital sequence information, this article provides commentary on the problem at hand. It substantiates the argument that rigidly interpreting the CBD to exclude digital sequence information, full-coding or not, from the meaning of genetic resources due to intangibility, would undermine the ABS regime. The article concludes by recapping key points of the discussion and highlighting the need for formal clarification on the meaning and scope of digital sequence information, and particularly, whether the ABS regime applies to it.An examination of the meaning, nature, and importance of digital sequence information in synthetic biology research is a useful starting point to provide the requisite technical grounding for the analysis of the concept and its potential impact on the ABS system.Study 1, which is one of the four studies commissioned by the CBD on digital sequence information on genetic resources pursuant to Decision 14/20, attempts to decompose the concept of digital sequence information into its constituent elements, “digital,” “sequence,” and “information.” Quoting the Oxford English Dictionary, the Study explains that “digital” speaks “of signals, information, or data: represented by a series of discrete values , typically for electronic storage or processing.” This means that digital connotes data held “in computer memory or data storage” .More challenging is the element, “information,” which may be distinguished from “data.” The latter describes the intrinsic properties of objects, while the former applies to the meaning or knowledge produced through “cognitive processes applied to such data” .Beyond the above effort, there is still no agreement on the precise meaning and scope of the concept of digital sequence information, introduced in Decisions CBD XIII/16 and Nagoya Protocol 2/14, which were respectively adopted at the thirteenth meeting of COP and the second meeting of COP-MOP in December 2016 . The study by Laird and others indicates that the uncertainty surrounding digital sequence information also permeates policy processes at the World Intellectual Property Organization (WIPO), the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), WHO’s Pandemic Influenza Preparedness (PIP) Framework, and the United Nations Convention on the Law of the Sea on the Conservation and Sustainable Use of Marine Biological Diversity of Areas beyond National Jurisdiction. The last three respectively use the alternative terms, “sequence data,” “resources in silico,” “digital sequence data,” and “genetic sequence data” .Between 2017 and 2018, parties to the CBD and the Nagoya Protocol, as well as other stakeholders, had the opportunity to express views on how uses of digital sequence information on genetic resources would likely impact the three objectives of the CBD .118Nevertheless, the report of the first (2018) AHTEG on digital sequence information on genetic resources attempts a classification of subject matter that may qualify as digital sequence information and be relevant to CBD objectives. This includes the following: “(a) nucleic acid sequence reads and the associated data; (b) information on the sequence assembly, its annotation and genetic mapping. This information may describe whole genomes, individual genes or fragments thereof, barcodes, organelle genomes, or single-nucleotide polymorphisms; (c) information on gene expression; (d) data on macromolecules and cellular metabolites; (e) information on ecological relationships, and abiotic factors of the environment; (f) function, such as behavioral data; (g) structure, including morphological data and phenotype; (h) information related to taxonomy; (i) modalities of use” .19The breath of the above classification further indicates the lack of consensus on the exact scope of digital sequence information .Study 2/3, another of the four CBD-commissioned studies, which focuses on traceability and databases, adopts a definition that combines (a) and (b) of AHTEG’s classification of digital sequence information presented above. It uses the term, “nucleotide sequence data,” and considers this to mean “ (a) nucleic acid sequence reads and the associated data” and “ (b) information on the sequence assembly, its annotation and genetic mapping” .With regard to the classification provided by Study 1 above, it is evident that each group is determined by the degree to which data or information is disconnected from the physical genetic resource. The nearer it is to the originating genetic resource, the narrower the scope of subject matter recognized as digital sequence information and vice versa. For example, Group 1, which is the closest to the originating genetic resource, has a narrow scope, covering only nucleotide sequence data relating to transcription. Group 2, which is intermediate in coverage, covers not only nucleotide, but also protein sequence data, that is, data and information relating to both transcription and translation. Group 3 is also intermediate in scope, but has a broader coverage than Group 2 because, in addition to nucleotide and protein sequence data, it also covers metabolites and biochemical pathways. Thus, it consists of information relating to not just transcription and translation, but also biosynthesis. Group 4, which is the farthest from, and least traceable or even non-traceable to the originating genetic resource, has the widest coverage that includes information concerning not only transcription, translation, and biosynthesis, but also “subsidiary information” like “behavioral data, information on ecological relationships and traditional knowledge” .Other terms, such as nucleotide sequence data, genetic resource sequence data, and genetic sequences used respectively by Study 2/3, the International Chamber of Commerce, and the PIP Framework, correspond with Group 1, which is the narrow category of digital sequence information proposed by Study 1. The March 2020 report of AHTEG on digital sequence information on genetic resources maintains that Groups 1–3 may be considered as digital sequence information, to the exclusion of Group 4, including traditional knowledge, which is viewed as “associated information,”With the narrowness of Group 1, it is much easier to trace the origin of digital sequence information; whether it is derived from or independently of the utilization of a tangible genetic resource , the dominant open access database for genetic sequences, are in the form of nucleotide sequence data .21Another issue may be raised here in respect of CRISPR  genome editing and how the CBD might deal with edited DNA; how easy it would be to trace an edited gene to the original version. A relevant question would be how similar DNA sequences have to be to their natural source so as to be recognized as having originated from states other than where they were edited, also bearing in mind that they may be modified into unnatural sequences through synthetic biology  contained in the nucleus of eukaryotes, how this is transcribed, then translated into proteins, and eventually biosynthesized into metabolites. The DNA comprises four nitrogenous bases called adenine, thymine, guanine, and cytosine , with the As being equal in number to the Ts, and the Gs to the Cs The DNA has two strands held together to form a double helix. Both strands comprise chemical subunits called nucleotides, each of which has a deoxyribose sugar, a phosphate group, and a nitrogenous base  .Also, based on the central dogma, the DNA regulates the creation of another type of nucleic acid called ribonucleic acid (RNA). The latter regulates the creation of proteins, which determine the structure of cells and the way they function .The mRNA carries that information to a ribosome in the cell’s cytoplasm where it is converted into an ordered chain of amino acids that form a protein . Concurrently, ease of access to sequencing technologies made possible by increasingly affordable costs soon inundated scientists with vast amounts of sequence data that proved too complex to handle. Bioinformatics and computational biology ameliorated this challenge by providing computational tools and platforms, including freely accessible databases that receive continually rising levels of DNA sequences and functional annotations, which together form what may broadly be considered as digital sequence information . Across the scientific and industrial fields, increasing numbers of technologies and applications that rely on digital sequence information address wide-ranging needs .Turmeric and Neem, among other cases.The CBD was adopted in 1992 during the Rio Earth Summit. Alongside the quest for the conservation and sustainable use of biodiversity, this instrument aims to ensure orderly access to genetic resources and the fair and equitable sharing of benefits arising from their utilization.Tumeric, a patent was granted on the use of the turmeric plant for healing wounds, an already established Indian traditional knowledge. India successfully petitioned the United States Patent and Trade Mark Office (USPTO) to nullify that patent States’ sovereignty over their biological resources and traditional knowledge, including the requirements of prior informed consent and equitable benefit-sharing on mutually agreed terms, are reiterated in the Nagoya Protocol.Since the ABS regime centers on control over “genetic resources”According to the CBD, “‘biological resources’ include genetic resources, organisms or parts thereof, populations, or any other biotic component of ecosystems with actual or potential use or value for humanity.”Over the years, that lack of precision has caused controversy over whether the international ABS regime applies only to genetic resources or extends to biological resources, as suggested by some domestic ABS laws . The second approach above could also undermine the ABS regime because resources accessed based on claims of non-genetic uses could later be put to genetic uses, thereby evading ABS obligations , where it defines a derivative as “a naturally occurring biochemical compound resulting from the genetic expression or metabolism of biological or genetic resources, even if it does not contain functional units of heredity.” Attempts to fill this gap rely on CBD’s negotiating history by suggesting that between 1989 and 1992 when the instrument was negotiated, the emphasis was on protein-coding sequences. Impliedly, “functional units of heredity” could only have been intended to mean full-coding sequences .It is also vital to examine other key elements in CBD’s definition of genetic resources and genetic material. According to Article 2(10) of the CBD, genetic resources should have “actual or potential value.” Neither the CBD nor the Nagoya Protocol explicitly defines this term. However, according to Preamble 1 of the CBD, such value may be “intrinsic, ecological, genetic, social, economic, scientific, educational, cultural, recreational and aesthetic,” which suggests that value does not only consist in genetic material  of the Nagoya Protocol, utilization of genetic resources “means to conduct research and development on the genetic and/or biochemical composition of genetic resources, including through the application of biotechnology as defined in Article 2 of the CBD.” According to Article 2 of the CBD and Article 2(d) of the Nagoya Protocol, biotechnology refers to “any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use.” Utilization, as defined here, is to be differentiated from non-ABS uses, such as the purchase and consumption of biological resources as commodities, for example, fruits, tubers, or vegetables .As already remarked, the Nagoya Protocol is meant to implement the CBD. It is unclear, however, whether the Protocol covers access to genetic resources occurring before or after 12 October 2014, when it became effective. Provider states adopt the former position, but user states favor the latter .On the other hand, treaty law apparently precludes the former position, which could be viewed as retroactive application of the Nagoya Protocol.43Interpretation is the task of ascertaining the actual meaning of a treaty, whereas application is the task of determining the repercussions visited by a treaty on the occurrence of specific facts, acts, or situations .In the context of the Nagoya Protocol, to determine its temporal scope, reference must be made to its text, subject matter and nature of obligations imposed. The Protocol’s objective is the fair and equitable sharing of benefits from the utilization of genetic resources.The Nagoya Protocol came into force 90 days after the date on which the fiftieth instrument of ratification was deposited,More importantly, for the present purposes, although the Nagoya Protocol can conveniently be said to exclude genetic resources accessed and utilized before 12 October 2014, it does not specifically address cases of genetic resources accessed before that date but utilized thereafter. This lack of clarity calls for interpretation, rather than application stricto sensu of the treaty. Interpretation is necessary to determine the intention of the parties as to its temporal scope. In this regard, it bears recalling that, according to Article 3 of the Nagoya Protocol, this Protocol applies to the genetic resources covered by Article 15 of the CBD. That provision of the CBD took effect as far back as 1993 and requires the prior informed consent of provider states before access to and utilization of their genetic resources. It also requires the sharing of results from research and development, as well as benefits accruing from commercial and other utilization of such resources. Therefore, provider states could argue that they are merely interpreting the Nagoya Protocol in a way that would best ensure its effectiveness, as well as that of the CBD, in light of their object and purpose (c) of the Vienna Convention, which views international law as a system. Therefore, it demands that, in applying a particular rule, other rules that are relevant to the matter at hand must also be considered  of the Nagoya Protocol permits parties to adopt appropriate legislative, administrative, or policy measures to ensure the fair and equitable sharing of benefits derived from the utilization of genetic resources, as required by this Protocol and the CBD. Thus, provider states could stipulate in their domestic ABS measures that all uses of genetic resources made after 12 October 2014 are covered, even if such resources were accessed before that date.49It is equally arguable that, strictly speaking, what is addressed by the Nagoya Protocol is not the accessing of genetic resources per se (act), which is generally permitted, subject to prior informed consent, but doing so in a manner adverse to the interest of providers; that is, the utilization of those resources, without fair and equitable benefit-sharing with providers (effect).53Accordingly, the issue as to retroactivity centers on whether the situation causing adverse effects, namely utilization without benefit-sharing, has ceased or still continues after the Nagoya Protocol is in force. Viewed in this sense, Article 28 of the Vienna Convention cannot be said to preclude benefit-sharing obligations on users in respect of genetic resources accessed before the Nagoya Protocol came into force, insofar as their utilization continues after that time. Since Article 28 of the Vienna Convention provides that a treaty does not apply to situations that have ceased to exist, unless a contrary intention is established, the converse is also true that the obligations of a treaty can be extended to situations that have not ceased to exist after the treaty is in force, even if the acts creating them started at a previous time.54Hence, a suggested strategy for overcoming the above dilemma is to subject all new uses of genetic resources occurring after the Nagoya Protocol’s effective date to benefit-sharing obligations, even if those resources were accessed before that date .The foregoing analysis throws up several interrelated questions that require fuller examination. Specifically, the following questions call attention: (i) given its merely informational, intangible nature, does digital sequence information qualify as genetic material and, therefore, as a genetic resource covered by the CBD and its ABS regime?; (ii) even if it does, could every such information, even if non-coding, be considered as “functional units of heredity”?; (iii) does digital sequence information have value?; (iv) is digital sequence information capable of utilization? and (v) does “access” to genetic resources occur only when there is interaction with a physical genetic material?.It is evident from the preceding discussion that the CBD defines “genetic resources,” “genetic material,” “derivative,” and even “biotechnology” all in material terms .In interpreting a treaty term, such as “functional units of heredity,” extrinsic aids, including negotiating histories, are useful for resolving uncertainties.Indeed, synthetic biology research is directed not only at the enhancement of protein-coding sequences, but also those performing other valuable functions. Through such research, parts of nucleic acid smaller than genes have been exchanged between species to synthesize functional gene products .Enhancement of sequences encoding proteins or other vital functions involves substantial utilization of physical genetic material . Emphasis on protein-coding sequences is untenable for other reasons. Genes are structurally mutable. Through “natural genetic engineering” encoded information could be altered by other molecules, such as proteins . Additionally, appreciation of DNA functionality and potential value is relative, being dependent on the current state of scientific knowledge .56 Perce57Phenomenal advances in the life sciences witnessed in recent decades point vividly to the ever present possibility that “research may identify new functions of genetic resources and streamline the development of technology, new goods and services” .As previously mentioned in this article, a genetic resource should have actual or potential value and, therefore, be capable of utilization. It should be recalled that utilization, as defined under Article 2(c) of the Nagoya Protocol, covers research and development exploiting the “genetic and/or biochemical composition of genetic resources including through the application of biotechnology.” It is tempting to confine “biotechnology,” as defined in that article, to technology applying tangible resources, rather than intangible information. However, the preceding phrase, “research and development on the genetic and/or biochemical composition of genetic resources” suggests that utilization of genetic resources can also occur through information repositories and dissemination of sequence information .It means that, as defined in the CBD, biotechnology is more expansive than genetic engineering and covers synthetic biology .Reinforcing the above view, Muller observes that biotechnological research is directed at the informational element of genetic resources  pAdditionally, a report by the Organization for Economic Cooperation and Development (OECD) suggests that “utilization” of genetic resources includes bioinformatics. This covers the construction of databases on genomes and protein sequences, as well as the modeling of complex biological processes, including through the use of systems biology . The OECIn the above regard, the EU’s position is that, until international agreement is reached on the issue by Parties to the Nagoya Protocol, the utilization of digital sequence information obtained from public databases is not covered by the Nagoya Protocol and related EU regulations,66The above arguments are consistent with other authoritative commentaries. In a report written by the Fridtjof Nansen Institute for the CBD, Schei and Tvedt maintain that bioinformatics provides the means for “realizing the value in the genetic material disconnected from the biological sources where it was originally found” .Although synthetic biology remains largely a “wet laboratory science” . Similarly, Article 5(1) of the Nagoya Protocol requires the fair and equitable sharing of benefits from the utilization of genetic resources, including “subsequent applications and commercialization.” This quotation has a potentially wide coverage that could also include uses of non-naturally occurring components, such as digital sequence information, given the Protocol’s definition of “utilization of genetic resources,” “derivatives,” and “biotechnology” .That phrase, “in the form of molecules and substances,” suggests that the object of regulation is tangible material, not intangible information, as ostensibly intended. The country’s new ABS legislationIn India, ABS measures include the Biological Diversity Act (BDA) and the Access and Benefit-Sharing Guidelines, both of which also allude to digital sequence information derived through access to biological resources, warranting benefit-sharing , p. 53).Nevertheless, some countries have expressly excluded digital sequence information from their domestic ABS regimes. For example, Japan’s ABS guidelines do not consider digital sequence information as falling within the meaning of genetic resources, and expressly exclude “information concerning genetic resources, such as nucleic acid base sequences.”Notably though, in both the Commonwealth and the Queensland territory of Australia, digital sequence information accessed from a database and used independently of a physical genetic resource to develop and commercialize a product, is not covered, unless expressly agreed at the time of access to the physical genetic resource .82In Association for Molecular Pathology et al. v Myriad Genetics et al.,It is evident from the foregoing review that, although some countries have expressly excluded digital sequence information from their ABS systems, several others have either expressly included it within the meaning of genetic resources in their ABS systems or adopted a broad interpretation of genetic resources in existing ABS regimes so as to cover it. Thus, access to digital sequence information is equated with access to a physical genetic resource, which imposes benefit-sharing obligations, given that the former is derived from the utilization of the latter. Overall, it can be concluded that digital sequence information, as “the bioinformatic decoding” of physical genetic resources clearly amounts to the utilization of such resources. Indeed, since genome sequencing is an indispensable enabler of synthetic biology, the latter can be seen as a benefit arising from the former. Consequently, the synthetic biology field, as a benefit, is appropriately subject to the CBD’s objective of ensuring the fair and equitable sharing of benefits arising from the utilization of genetic resources through sequencing processes. This provides a sound rationale to extend the reach of the CBD and the Nagoya Protocol to the use of digital sequence information as a key resource in the synthetic biology field.In the above regard, it is vital to recall that Decision XIII/17Scholarship on the applicability of the ABS regime to digital sequence information mostly engages with the CBD, the Nagoya Protocol, and domestic ABS provisions. However, as a further contribution to the scholarly heritage, this article incorporates treaty interpretation and case law. Doing so significantly guides the interpretation of the contested terms, “genetic resources” and “functional units of heredity.” As treaties, the CBD and the Nagoya Protocol come within the realm of public international law. A pertinent question is how they should be interpreted amid uncertainty. The CBD provides that disputes over its interpretation or application shall be resolved through negotiation,Despite their acknowledged merits, those alternative dispute resolution methods have limited value for the present purposes. Generally, the outcomes of negotiation or conciliation proceedings do not bind the parties concerned. Arbitral awards are binding, but only on the parties to the arbitration proceedings. Hence, they have limited precedential value. Consequently, arbitral tribunals presiding over disputes with substantially similar facts and legal questions may make directly opposite awards.Under international law, treaty interpretation is governed by customary international law as the established practices among states; the Vienna Convention; judicial decisions; and the teachings of the most highly qualified publicists.Yet, to remain relevant, laws must metamorphose with changes in the times and circumstances of their adoption Evolutionary interpretation of a treaty term may also be justified by the requirement in Article 31 of the Vienna Convention to interpret a treaty in good faith, consistent with its object and purpose, taking into account relevant international law rules .Dispute Regarding Navigational and Related Rights,107In Aegean Sea, the ICJ similarly ruled that the generic term, “territorial status,” used in an instrument intended to have a “continuing duration,”Conclusions on the Fragmentation of International Law clarifies that, based on Article 31(3)(c) of the Vienna Convention, this new meaning of a generic term shall be determined from the international law applicable in relations between the parties at the time of the treaty’s application .tivity” tivity” s scope United States-Import Prohibition of Certain Shrimp and Shrimp Products,Decisions of quasi-judicial bodies similarly reflect evolutionary treaty interpretation. In Instructively, the Appellate Body emphasized that, although the GATT language, “resources,” was used over 50 years ago and subsequently transplanted in an unmodified form into the WTO Agreements in 1994, that term was not static in reference, but “by definition evolutionary.” Namibia legal Consequences, as well as the United Nations Convention on the Law of the Sea (UNCLOS) 1992,118In interpreting “exhaustible resources” to include living and non−living resources, the Appellate Body also referred toConclusions on the Fragmentation of International Law, the International Law Commission similarly maintains that a treaty term may be presumed to have been intended to have an evolutionary meaning where it requires taking into account subsequent changes of technical, legal, or economic nature or where that term is of such a general character as to necessitate taking into consideration changing situations  is not ‘static’ in its content or reference, but is rather ‘by definition evolutionary.’”In its uations .Altogether, the real essence of evolutionary interpretation is to ensure that a treaty effectively achieves its object and purpose, having regard to changes in its circumstances. Thus, in Iron Rhine Arbitration,123From the foregoing analysis, the following conclusions can be drawn with regard to the CBD. “Genetic resources” and “functional units of heredity” are scientific terms. Also, they are generic in that they merely refer to an indefinite category of referents. Further, they are vague because, as argued earlier, despite the references remaining the same, there is potential for their scope to evolve with changing times. Based on ICJ’s jurisprudence, such terms must, by definition, be deemed evolutionary. Even if this contention fails to hold, those terms would still warrant evolutionary interpretation based on the ICJ’s general rule. Not only are they generic, but also, the relevant treaties in which they are used, the CBD and the Nagoya Protocol, have continuing durations. It is, therefore, reasonable to presume an intention by the parties thereto that both texts should evolve with those changing references, thus justifying evolutionary interpretation of both terms.United States-Import Prohibition of Certain Shrimp and Shrimp Products shows that the term, “resources,” applies not only to living, but also non-living things, such as minerals and, analogously, digital sequence information .Despite controversies over whether a treaty may have only one or several objects and purposes . As a whole, the INSDC has users from every country in the world. Most of them are, however, from developed countries, although Brazil, India, and Singapore are also emerging .The INSDC databases contain a metadata tag for “/country,” which permits depositors to report the country of origin of nucleotide sequence data, but, as Study 2/3 observes, this term does not perfectly correspond to “country of origin.” Even at that, reporting of country of origin of INSDC entries remains poor. Despite a gradual increase in reporting, in 2018, barely over 40% of nucleotide sequence data deposited indicated a country of origin, which suggests that potentially thousands to millions of sequences do not carry country of origin information . Surely, open access to digital sequence information is supportable and consistent with CBD requirements, particularly with regard to biodiversity conservation  crisis, for example, Indonesian strain of the virus shared by the country with WHO’s Global Influenza Surveillance Network, was accessed and used by foreign pharmaceutical firms to develop vaccines. Those vaccines were patented and sold at prices beyond the reach of afflicted Indonesians, leaving scores of them dead , 99)..99]).States like Japan and Costa Rica view open access to digital sequence information derived from physical genetic resources as a non-monetary form of benefit-sharing; they support the deposit of sequence data in openly accessible databases to promote scientific development .129Additionally, the INSDC partner institution, GenBank, allows depositors to claim intellectual property rights, including patents, in all or aspects of deposited data ,It is worth recalling that, in establishing the international ABS regime, CBD parties sought to ensure fair and equitable sharing of benefits from the utilization of genetic resources so as to promote access to sustainable use and conservation of biodiversity. To effectively pursue these goals and fully achieve the CBD’s object and purpose, “genetic resources” and “functional units of heredity” should be construed in such a dynamic manner as would include digital sequence information , It is untenable that the genetic composition of a genetic resource utilized in such a way, even if transformed through biotechnology, should be excluded from the ABS regime, simply because it was accessed digitally or is non-coding. To give “genetic resources” its original, restrictive meaning would rob the ABS regime of the potential value of genetic material utilized in synthetic biology or other future biotechnologies .This article has examined the applicability of the CBD and its ABS regime to digital sequence information on genetic resources. The CBD’s original wording apparently conceives genetic resources in material terms, and some observers view “functional units of heredity” as full-coding sequences, even though undefined by the CBD and the Nagoya Protocol. Nevertheless, relying on expert reports, studies, the CBD, the Nagoya Protocol and domestic, as well as regional ABS legislation, this article has argued that rigidly interpreting the CBD to exclude digital sequence information, full-coding or not, would weaken the ABS regime.To remain relevant and achieve its objectives, the CBD must evolve with scientific progress. As the sequencing of regular genetic resources and open dissemination of sequence information expands, the potential for inappropriate access to the genetic resources of provider states would likely increase. This makes it appropriate to adopt a dynamic interpretation of the CBD, which views digital sequence information, full-coding or not, in the same way as their physical manifestations. In reinforcing these arguments, this article invoked evolutionary interpretation as a veritable tool for aligning the CBD and the Nagoya Protocol with the changes that have followed their adoption.Treaties cannot anticipate all future possibilities and are usually drafted in general terms. Apart from the present, they may eventually have to address completely different future situations. However, once adopted, they remain static, despite changes in the scientific landscape that influenced their drafting. Article 29 of the CBD permits treaty amendment, but forging international consensus to amend treaties and reconcile them with new realities is difficult. Yet, to remain relevant, they must evolve with changing circumstances. Evolutionary interpretation helps in meeting this challenge.Ultimately though, formal clarification is needed on the precise meaning of digital sequence information, its scope, whether narrow, broad or intermediate, and particularly, whether it is covered by the ABS regime. As well, ideal methods of tracing the generation and utilization of digital sequence information, identifying beneficiaries, and sharing benefits need to be settled, as is the distinction between data and information, which could be vital in determining the coverage of the concept of digital sequence information. Public databases, particularly the INSDC, could strive to facilitate traceability through more diligent country of origin reporting for environment-related sequence data submissions. In this regard, their involvement in CBD deliberations would be salient. The tracking of Internet Protocol addresses and use of blockchain system to monitor uses of sequences could be explored, together with likely challenges. Also, capacity building for developing provider states and the possibility of establishing a Global Multilateral Benefit-Sharing Mechanism suggested under Article 10 of the Nagoya Protocol could be considered as a way of ensuring fairer and more equitable benefit-sharing.It is expected that Part 2 of COP-15 would make progress on the above matters. This would enhance hopes for the realization of ABS objectives and provide certainty for users of genetic sequences as to the scope of potential ABS obligations facing them. For now, researchers, including those from a jurisdiction, such as the USA, which is not subject to the CBD and the Nagoya Protocol, should note that they may be bound by domestic or regional ABS laws. Violations may incur penal sanctions or denial of valuable intellectual property in jurisdictions where protection is sought. It is prudent to seek information and advice from relevant sources, including National Focal Points, Competent National Authorities and the Access and Benefit-sharing Clearing House. Further developments, particularly the outcomes of COP meetings, should be monitored closely.


Genetic parameters for daily predicted gross feed efficiency (pGFE) and energy corrected milk (ECM) in the first three parities of South African Holstein cattle were estimated by repeatability animal models. Data comprised of 11,068 test-day milk production records of 1,575 Holstein cows that calved between 2009 and 2019. Heritability estimates for pGFE were 0.12 ± 0.06, 0.09 ± 0.04 and 0.18 ± 0.05 in early, mid and late lactation, respectively. Estimates were moderate for primiparous (0.21 ± 0.05) and low for multiparous (0.10 ± 0.04) cows. Heritability and repeatability across all lactations were 0.14 ± 0.03 and 0.37 ± 0.03, respectively. Genetic correlations between pGFE in different stages of lactation ranged from 0.87 ± 0.24 (early and mid) to 0.97 ± 0.28 (early and late), while a strong genetic correlation (0.90 ± 0.03) was found between pGFE and ECM, across all lactations. The low to moderate heritability estimates for pGFE suggest potential for genetic improvement of the trait through selection, albeit with a modest accuracy of selection. The high genetic correlation of pGFE with ECM may, however, assist to improve accuracy of selection for feed efficiency by including both traits in multi-trait analyses. These genetic parameters may be used to estimate breeding values for pGFE, which will enable the trait to be incorporated in the breeding objective for South African Holstein cattle. The dairy industry is increasingly under pressure to improve feed efficiency, due to the need to maintain herd profitability in an era of increasing feed costs, as well as growing concerns to safeguard the environment Connor . Gross fDry matter intake is a major component of feed efficiency traits, including GFE Connor, . Direct Due to scarcity of data on feed intake, there is a paucity of information on the genetic attributes of GFE in dairy cows  R2 = 0.79; root mean squared error (RMSE) = 1.05 kg/day), while a model that comprised butterfat yield, MY and LW had the highest ability to predict GFE  . Records for the period 2009 to 2019 were considered.Test-day records and pedigree data of cows from nine intensively-fed Holstein herds, participating in the South African National Milk Recording and Improvement Scheme, were obtained from the Integrated Registration and Genetic Information System of South Africa (Energy-corrected milk (ECM) yield (kg/day) was calculated from test-day milk yield (kg/day), protein (%), butterfat (%) and lactose percent (%), using Eq. R2) of 0.87 and 0.80, respectively. Gross feed efficiency was calculated as the ratio between ECM (kg/day) and DMI (kg/day).Prediction models for GFE were developed prior to this study, for primiparous . Non-genetic factors influencing pGFE and ECM, which required to be fitted in the models for (co)variance components estimation, were also determined using the General Linear Models procedure of the Statistical Analysis System . These effects included age of cow at calving, lactation stage, parity and herd-test-day.Bivariate analyses were carried out to estimate (co)variance components, as well as heritability and repeatability, for pGFE and ECM, within stages of lactation, for the entire first lactation (primiparous), entire second and third lactations (multiparous), and all entire lactations pooled together. All the analyses were conducted by the restricted maximum likelihood (REML) procedure of the ASReml 4.2 software  variance structure for random effects in the models was as follows:Genetic trend for pGFE, for all the lactations combined, was estimated by plotting average estimated breeding values (EBVs) by year of birth. The EBVs were estimated by the Best Linear Unbiased Prediction method (Henderson Means and coefficients of variation (CV) for daily pGFE and ECM, by stage of lactation and for entire-lactations, are presented in Table Estimates of heritability for daily pGFE and ECM within stages of lactation are shown in Table Table Estimates of genetic and phenotypic correlations among daily pGFE in different stages of lactation are presented in Table The genetic correlation between pGFE in primiparous and multiparous cows was strong and positive (0.99 ± 0.21). On the other hand, the phenotypic correlation was low (0.27 ± 0.03).Table Genetic trend for daily pGFE, for cows born between 2007 and 2017, is presented in Fig. Gross feed efficiency (GFE) is an exceptionally important trait in dairy production, due to its impact on profitability and environmental sustainability. Energy-corrected milk is a major predictor trait for GFE, and is also of great economic importance. Besides genetic variation between populations, differences in measurement or prediction methods may account for discrepancies in mean GFE among studies . It means pGFE in primiparous and multiparous cows may be under the influence of the same or linked genes. The observed correlation suggest that selection for pGFE based on first lactation data will result in improvement in later lactations. Such an approach is further justified by our finding that the heritability of pGFE is higher in primiparous than multiparous cows.Knowledge of the genetic correlations between pGFE and ECM may assist in improving accuracy of selection of pGFE, as well as incorporating it in the selection objective. These estimates were all positive and substantially high (> 0.90), within stages of lactation and across parities, confirming earlier findings by Köck et al.  who repoRepeatability was estimated to assess the extent to which repeated measures of pGFE and ECM across lactations are under the influence of permanent effects. Repeatability was moderate (0.37 ± 0.01 to 0.52 ± 0.02) for both pGFE and ECM, in agreement with a previous study by Köck et al.  on AustrGenetic trend for pGFE, across all lactations, was ascertained to assess if there have been any changes in genetic merit for the trait in recent years, in the South African Holstein cattle population. Such a change may occur as a correlated response to selection for other traits with which it is genetically correlated. There has been sustained genetic selection for yield traits in the South African Holstein cattle population (Ramatsoma et al. Results of this study indicate that daily gross feed efficiency predicted from milk components exhibits modest genetic variation, with the highest heritability being in first-parity, and in late lactation. High genetic correlations among pGFE in different stages of lactation indicate that records of pGFE along the lactation trajectory can be considered as repeated measures of the same trait. Hence, selection for pGFE based on late lactation records only seems reasonable, as it would achieve the highest accuracy of selection while improving the trait across the whole lactation. Higher heritability of pGFE in primiparous compared to multiparous cows, coupled with high genetic correlations between these lactations, justifies selection on first lactation records only. There appears to be scope for improving accuracy of selection for feed efficiency through multiple-trait analysis including ECM, due to the high genetic correlation between the two traits. Genetic trends show a slight increase in the genetic merit for pGFE in South African Holstein cattle in recent years, which may be a correlated response to selection for higher milk yield or other correlated traits. There is, however, a need to achieve significant improvement in the genetic merit of feed efficiency, in order to achieve profitable and environmentally sustainable dairy production systems. The genetic parameters obtained in the current study can be applied to estimate EBVs for pGFE, which may be used to achieve such improvement. Further enhancements to the selection programme could be effected through the application of random regression modelling, as well as identification of markers or genes influencing feed efficiency.