The successful use of Assisted Reproductive Technologies (ARTs) such as artificial insemination (AI) in small ruminants and other mammals depends on many factors, the most important of which is the quality of the semen used. The liquid storage and cryopreservation of spermatozoa at lower temperatures are associated with artificial insemination and rapid genetic improvement programs in the sheep production industry. Several studies have reported increased lipid peroxidation (LPO) and highly reactive oxygen species (ROS) production during liquid storage or cryopreservation of ram semen, leading to oxidative stress (OS), decreased antioxidant defense, and changes in sperm quality parameters like biokinetic and biochemical characteristics, viability, functional membrane and DNA integrity, along with mitochondrial activity. Therefore, supplementing ram semen extenders with exogenous antioxidants before preservation could mitigate this harmful effect. Various in vitro studies have reported improvements in ram sperm quality parameters like motility indexes, vitality, functional membrane/DNA integrity, antioxidant enzyme activity, total antioxidant content, mitochondrial activity, in vivo/in vitro fertility with significant decline in sperm abnormality, free radical production, LPO, ROS, apoptosis rate, and cytochrome C release from the mitochondrial matrix after the addition of various natural and synthetic antioxidant (vitamins, glutathione, taurine, pyruvate, melatonin, cysteine, selenium, zinc, plant extracts, sugars, amino acids, polyphenols) substances during preservation. Therefore, this review summarizes recent findings on oxidative stress-induced damage to sperm quality parameters in various ram breeds during chilling storage and cryopreservation. Moreover, supplementing basic semen extenders with different non-enzymatic antioxidant substances as a method to maintain sperm quality—along with their efficacy in reducing or preventing sperm damage during preservation—was discussed in detail.

Semen cryopreservation is associated with cryo-damages that compromises sperm quality parameters post-thawing. Hence, the present study was planned to evaluate the influence of supplementation of puerarin (PUE) to Tris-based extender on post-thaw semen quality parameters of ram semen, with a view to determine the optimal puerarin concentration for the cryopreservation of Hu ram semen. For this purpose, 100 ejaculates collected twice weekly from five Hu rams were used. Ejaculates collected from 5 rams on each collection day were pooled to yield 20 pooled samples for further processing. These samples were diluted with a Tris-based extender supplemented with 0, 25, 50, 75 and 100 & micro;mol/L of PUE solution in DMSO, cryopreserved and stored in liquid nitrogen. Post-thaw results showed that the addition of 75 & micro;mol/L PUE significantly improved total sperm motility, progressive sperm motility, as well as some bio-kinetic characteristics, compared with the control and other PUE supplementation groups (P<0.05). Sperm bio-kinetic characteristics including amplitude of lateral head displacement, angular displacement and wobble in PUE 75 and 100 & micro;mol/L supplementations were also higher than those of the control group (P<0.05). Moreover, sperm acrosome integrity, plasma membrane integrity, along with semen total antioxidant content, activities of catalase and superoxide dismutase enzymes, and mitochondrial membrane potential were correspondingly higher in the PUE 75 & micro;mol/L group post-thaw (P<0.05). In addition, PUE 75 & micro;mol/L treatment significantly decreased semen ROS and malondialdehyde level (P<0.05). In-vitro fertilization rate of semen in the PUE 75 & micro;mol/L group (57.14%) was higher than 38.00% for the control group (P<0.05). In conclusion, supplementation of 75 & micro;mol/L PUE to a Tris-based extender was considered optimal, as it improved post-thaw sperm motility indexes, bio-kinetic characteristics, mitochondrial function, antioxidant enzymes activity, and fertilization potential of Hu ram semen.

Hu sheep are a well-known Chinese dual-purpose breed valued for meat production, early sexual maturity, high prolificacy, and adaptability to hot, humid environments. In this study, we combined whole-genome sequencing, GWAS, and eQTL analyses to investigate the genetic basis of growth and slaughter traits in 420 eight-month-old Hu sheep. Using a discovery cohort (N = 112) and a validation cohort (N = 308), we integrated high-throughput SNP genotyping and RNA sequencing, identifying 559,996 high-quality SNPs and multiple significant loci associated with traits such as live weight and carcass weight. Among the 2,368 cis-eQTLs detected, the most significant was linked to ZNF280B expression in the longissimus lumborum muscle. Functional validation showed that ZNF280B downregulation significantly inhibited skeletal muscle satellite cell proliferation and induced apoptosis, highlighting its critical role in muscle development and fat metabolism. The novelty of this study lies in its large sample size, comprehensive multi-trait analysis, and the integration of functional validation, providing reliable genetic markers for marker-assisted selection in Hu sheep. These findings deepen our understanding of the genetic mechanisms underlying growth and slaughter performance and offer valuable insights for improving production efficiency and promoting sustainable livestock development.

Ram sperm are very susceptible to oxidative stress damage during low-temperature preservation. Seminal oxidative stress results in membrane lipid peroxidation, changes in the normal physiological and enzymatic pathways, finally associated with spermatozoa motility loss, reduced fertility and compromised embryogenesis. Lycopene is one of the richest natural sources of antioxidants. The effects of lycopene (LYC) supplementation of the semen diluent upon the quality of Hu ram semen preserved for five days at 4 degrees C were investigated in this study. Semen ejaculates collected from 5 rams twice weekly for 12 weeks were pooled on each collection day, diluted with an extender supplemented with 0.5, 1.5, 2.5, and 3.5 mu M of LYC, while the control group received no supplementation. Sperm viability, motility variables, and membrane integrity were evaluated daily up to five days. Semen biochemical indexes, including oxidation status, mitochondrial potential, and antioxidant activity, were assessed on the fifth day of storage. The results showed that supplementation of LYC at 2.5 mu M significantly (P <= 0.05) increased the sperm viability, sperm total and progressive motility percentages, sperm plasma membrane integrity, and acrosome integrity during preservation at 4 degrees C. It also significantly (P <= 0.05) enhanced the total antioxidants content and antioxidant enzymes activities like catalase and superoxide dismutase. The mitochondrial membrane potential of sperm was correspondingly increased during preservation. However, supplementation of LYC at 2.5 mu M to the extender significantly (P <= 0.05) reduced highly detrimental free reactive oxygen species production and malondialdehyde contents. In conclusion, the present study showed that supplementation of the extender with 2.5 mu M LYC significantly recovers the quality of sperm by improving the antioxidant enzyme activity and mitochondrial function, decreasing the damage caused due to free radicals, lipid peroxidation (MDA), and oxidative stress (ROS) to Hu rams' semen.

OBJECTIVE:Hair morphogenesis is tightly related to hair follicle stem cells (HFSCs) proliferation and hair follicle (HF) development. Yangtze River Delta white goats (YRDWG) HFSCs are important for producing superior-quality brush hair (SQBH). Nonetheless, the known regulatory mechanisms are not sufficient to explain YRDWG gHFSCs growth, HF development, and SQBH formation. METHODS:To deeply investigate the interaction networks and mechanisms of circCOL1A1 in the HF development and SQBH formation of YRDWG in detail, we applied whole-transcriptome sequencing and bioinformatics analysis of circCOL1A1-knockdown and circCOL1A1-overexpressing HFSCs from YRDWG. STRING and other databases were used to construct multiple interaction networks. Differentially expressed (DE) genes, DE-miRNAs, and DE-circRNAs were further confirmed via real-time quantitative polymerase chain reaction and Sanger sequencing. RESULTS:A total of 87 genes, 96 miRNAs, and 135 circRNAs were DE between circCOL1A1-knockdown and circCOL1A1-overexpressing gHFSCs. Functional enrichment, gene ontology annotation and Kyoto encyclopedia of genes and genomes analyses identified marked enrichment of these DE- genes, DE-miRNAs, and DE-circRNAs in the MAPK, PI3K/Akt, and focal adhesion signaling pathways, which are closely associated with gHFSCs growth and HF development. In addition, through interaction network construction, four important regulatory axes were obtained, namely, the chi-circCOL1A1-miR-149-5p-CMTM3-AR, chi-circACTN1- miR-671-5p-MAPK3/COL13A1, chi-circITGA6-miR-18a-5p-FGF1/MAP3K1 and chi-circ COBLL1-miR-30a-5p/miR-128-3p-ITGA6/MAPK14/FGF14 axes. CONCLUSION:These novel findings provide a valuable and comprehensive basis for investigating the complex mechanism by which circRNAs participate in and regulate HF development and SQBH formation in YRDWG.

The effect of β-Carotene on ram semen cryopreservation has not been previously evaluated. The present research aimed to investigate the impact of different concentrations of β-Carotene in the diluent on the quality of ram semen and to analyze its antioxidant effects by the addition of tert-butyl hydroperoxide (TBHP) during cryopreservation. Semen samples were diluted with a Tris-based extender containing the β-Carotene (0, 10, 20, 30 and 40 mg/L). The motility and biokinetic characteristics, membrane and acrosome integrity, related parameters of oxidative stress, mitochondrial membrane potential (MMP) and apoptosis rate were measured after the cryo-preservation. The results indicated that the 20 mg/L β-Carotene group significantly improved total motility (TM), progressive motility (PM), average motion degree (MAD), membrane and acrosome integrity, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) compared to the other groups (P < 0.05). Additionally, this group significantly reduced reactive oxygen species (ROS) and malondialdehyde (MDA) content compared to the control group (P < 0.05). Furthermore, the TM, PM, wobble (WOB), MAD, membrane and acrosome integrity, CAT, SOD, T-AOC and MMP in the 100 μM TBHP+20 mg/ml β-Carotene group were higher than those in the 100 μM TBHP group (P < 0.05). The combined supplementation group also significantly decreased the ROS, MDA, the protein level of Cytochrome C and sperm apoptosis rates (P < 0.05). Therefore, 20 mg/L is identified as the optimal concentration for the cryopreservation of ram semen, and β-Carotene can improve the oxidative stress damage during the frozen-thawed process by enhancing the antioxidant capacity of sperm and maintaining mitochondrial function.

Lamb is highly regarded worldwide for its unique flavor, rich nutritional value, and diverse cooking adaptability. This study aimed to characterize the dynamic changes in volatile metabolites during post-mortem maturation of Hu sheep meat. Volatile metabolites in the longissimus thoracis muscles of eight 8-month-old male Hu sheep were profiled at 6, 12, 24, 48, 72, and 96 h post-slaughter using gas chromatography-mass spectrometry (GCMS). The results revealed that 198 volatile metabolites were identified in muscle samples of Hu sheep meat at different time points during post-mortem maturation. A total of 36 key volatile metabolites were screened based on the criteria of Variable Importance in the Projection (VIP) >1, P <0.05, and Fold Change (FC) >2 or <0.5. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) revealed significant alterations in volatile metabolites at 48 h post-slaughter during the post-mortem maturation of Hu sheep meat. Furthermore, key metabolic pathways were identified from The Kyoto Encyclopedia of Genes and Genomes (KEGG) database, including arginine and proline metabolism, steroid biosynthesis, and nitrogen metabolism, which were found to play critical roles in shaping the flavor profile of lamb during post-slaughter maturation. These findings offer novel insights into the meat quality changes of Hu sheep meat throughout post-mortem maturation.

The objective of this research was to investigate the effect of astaxanthin supplementations of semen extender on the quality of Hu ram semen after up to five days of preservation at 4 °C. Semen samples were collected from five healthy Hu rams using an artificial vagina during breeding season (April to August 2023) and diluted with a basic extender supplemented with control (0), 1 µM, 2 µM, 3.5 µM, or 4.5 µM of AXT. Overall, 170 semen ejaculate samples (34 repetitions) from five healthy Hu rams were used in our research study. The results revealed that the addition of AXT (3.5 µM) significantly (p ≤ 0.05) increased the sperm kinematic indexes (T.M%, P.M%, MAD%, STR%, and LIN %), sperm viability, plasma membrane integrity, acrosome integrity, total antioxidant content (T-AOC), and mitochondrial membrane potential (MMP) of the Hu rams spermatozoa after up to five days of preservation at 4 °C. Contrary to that, the addition of the best concentration of AXT (3.5 µM) to the semen extender significantly (p ≤ 0.05) reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) concentration of Hu ram semen. In conclusion, the results of the current study indicate that the addition of a semen extender with AXT improves the quality of Hu ram spermatozoa by increasing the total antioxidant capacity (T-AOC) and mitochondrial membrane potential (MMP). On the other hand, reducing free radicals induced oxidative (ROS) and per oxidative (MDA) damage to Hu ram semen.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

OBJECTIVE:The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. METHODS:In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide, dimethyl sulfoxide, different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity, curvilinear velocity, average path velocity, amplitude of lateral head displacement, wobble movement coefficient, average motion degree, functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. RESULTS:When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p<0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p<0.05) decreased the ROS level of sperm. In this study, the TM, PM, and membrane integrity of the 6% EG were significantly (p<0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p<0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. CONCLUSION:In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

IntroductionRam spermatozoa inevitably produce a large number of reactive oxygen species (ROS) during liquid storage, leading to oxidative stress and a decline of spermatozoa quality. Therefore, it is particularly important to add exogenous antioxidants during the process of semen liquid preservation. The purpose of this study is to investigate whether adding alpha-lipoic acid (ALA) to ram semen can reduce oxidative stress and enhance spermatozoa quality during the liquid storage at 4°C.MethodsDifferent concentrations of ALA (0, 0.025, 0.05, 0.1, 0.5, 1 mM) were added to semen and stored at 4°C. During storage at 4°C, spermatozoa motility, kinetic parameters, membrane integrity, acrosome integrity, energy metabolism parameters (mitochondrial membrane potential (ΔΨM) and adenosine triphosphate (ATP)) and oxidative stress parameters [ROS, malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD)] were assessed.Results and discussionThe results indicated that 0.1 mM ALA significantly (p<0.05) improved spermatozoa total motility (TM) and progressive motility (PM), plasma membrane integrity, acrosome integrity, ΔΨM, ATP, TAC, and SOD, while significantly (p<0.05) reducing spermatozoa ROS and MDA content compared to the control group. In conclusion, ALA can reduce damage caused by oxidative stress in spermatozoa and effectively improve the quality of semen preserved at 4°C. And the optimal concentration is 0.1 mM.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

OBJECTIVE:Hair follicle stem cells (HFSCs) differentiation is a critical physiological progress in skin hair follicle (HF) formation. Goat HFSCs differentiation is one of the essential processes of superior-quality brush hair (SQBH) synthesis. However, knowledge regarding the functions and roles of miR-133a-3p and miR-145-5p in differentiated goat HFSCs is limited.METHODS:To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p (Mimics and Inhibitors) separately or combined were performed. NANOG, SOX9, and stem cell differentiated markers (β-catenin, C-myc, Keratin 6 [KRT6]) expression levels were detected and analyzed by using real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence assays in differentiated goat HFSCs.RESULTS:miR-133a-3p and miR-145-5p inhibit NANOG (a gene recognized in keeping and maintaining the totipotency of embryonic stem cells) expression and promote SOX9 (an important stem cell transcription factor) expression in differentiated stem cells. Functional studies showed that miR-133a-3p and miR-145-5p individually or together overexpression can facilitate goat HFSCs differentiation, whereas suppressing miR-133a-3p and miR-145-5p or both inhibiting can inhibit goat HFSCs differentiation.CONCLUSION:These findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings for further investigating goat hair follicle development.

Meat and meat products have a critical role in the human diet as important high-nutrient foods that are widely consumed worldwide. This study evaluated the effects of postmortem aging on Hu sheep’s meat quality in the longissimus dorsi (LD) muscle during postmortem aging. The samples were stored at 4 ± 1 °C; the meat quality was measured at 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h of postmortem aging. The results showed that, during the postmortem aging process, the pH of the muscles first decreased and then increased, and the shear force first increased and then decreased. The muscle fiber skeleton began to degrade, and the overall meat quality was improved to some extent. In addition, through ACQUITY UPLC I-Class Plus IMS Qtof identification of the muscle samples at different time points during the postmortem maturation process of the meat of Hu sheep, a total of 2168 metabolites were identified, and 470 metabolites were screened based on the VIP, P, and FC values, of which 79 were involved in KEGG pathways. In addition, pathways such as sphingolipid metabolism, glycerophospholipid metabolism, phenylalanine metabolism, and fatty acid elongation and degradation play an important role in the metabolic product changes in the meat of Hu sheep throughout the entire maturation process. These findings provide some insights into the changes in meat quality during the post-slaughter maturation process of lake lamb.

The aim of this study was to investigate the effect of punicalagin, an antioxidant, on ram sperm quality. Semen samples were collected and pooled from five rams, then diluted using a Tris-based diluent containing various concentrations (0, 5, 15, 30 and 45 μM) of punicalagin. Sperm motility, plasma membrane integrity, acrosome integrity, total antioxidant capacity (TAC), reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP), superoxide dismutase (SOD) and catalase (CAT) were measured and analyzed during liquid storage at 4 °C. The results showed that the Tris-based solution containing punicalagin improved sperm motility, plasma membrane integrity, acrosome integrity, TAC, SOD, CAT and MMP, and decreased ROS content and MDA content. At the same time, the semen sample diluted with the Tris-based solution supplemented with 30 μM punicalagin achieved the best effect. The sperm total motility, progressive motility, plasma membrane integrity, acrosome integrity, TAC, SOD, CAT and MMP of the group supplemented with 30 μM punicalagin were significantly (p < 0.05) higher than those of the other groups on the 5th day during the liquid storage at 4 °C. Meanwhile, the ROS content and MDA content were significantly (p < 0.05) lower than those in the other groups. In conclusion, the optimal concentration of punicalagin in the Hu ram semen diluent was determined to be 30 μM. The results indicated that a diluent supplemented with punicalagin could enhance the quality of ram sperm preserved at 4 °C by increasing antioxidant capacity, mitochondrial potential and reducing oxidative stress.

The purpose of this study was to investigate the effects of different diluents and freezing methods on the quality of thawed sperm after cryopreservation and find an inexpensive and practical method for freezing Hu ram semen for use in inseminations under farm conditions. Ejaculates were collected from five Hu rams. In experiment I, ejaculates were diluted with eight different freezing diluents (basic diluents A, B, C, D, E, F, G, and H). After dilution and cooling, the samples were loaded into 0.25 mL straws and frozen using the liquid nitrogen fumigation method. In experiment II, diluent C was used as the basic diluent and the semen was frozen using liquid nitrogen fumigation and two program-controlled cooling methods. For analysis, frozen samples were evaluated in terms of motility parameters (total motility (TM), progressive motility (PM)), biokinetic characteristics (straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), wobble movement coefficient (WOB), average motion degree (MAD)), reactive oxygen species (ROS) level, and membrane and acrosome integrity. In experiment I, diluent C had higher TM, PM, and acrosome and membrane integrity and lower ROS compared to other extenders (p < 0.05) except diluent A. Diluent C exhibited higher (p < 0.05) VCL, VAP, ALH, WOB, and MAD compared to diluents B, D, E, and F. In experiment II, TM and all biokinetic characteristics did not show significant differences (p > 0.05) amongst the three freezing methods. Liquid nitrogen fumigation resulted in higher (p < 0.05) PM, membrane integrity, acrosome integrity, and lower ROS level compared to the program. In conclusion, the thawed semen diluted with diluent C had higher quality compared to other diluents. The liquid nitrogen fumigation demonstrated superior semen cryopreservation effects compared to the program-controlled cooling method using diluent C.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

AbstractHu sheep are a unique breed in our country with great reproductive potential, the extent of whose breeding has been steadily rising in recent years. The study subjects in this experiment were 8-month-old Hu sheep (n = 112). First of all, the growth performance, slaughter performance and meat quality of their eye muscle quality were assessed, meanwhile their live weight, carcass weight, body length, body height, chest circumference, chest depth and tube circumference were respectively 33.81 ± 5.47 kg, 17.43 ± 3.21 kg, 60.36 ± 4.41 cm, 63.25 ± 3.88 cm, 72.03 ± 5.02 cm, 30.70 ± 2.32 cm and 7.36 ± 0.56 cm, with a significant difference between rams and ewes (P < 0.01). Following that, transcriptome sequencing was done, and candidate genes related to growth performance were identified using the weighted co-expression network analysis (WGCNA) approach, which was used to identified 15 modules, with the turquoise and blue modules having the strongest association with growth and slaughter performance, respectively. We discovered hub genes such as ARHGAP31, EPS8, AKT3, EPN1, PACS2, KIF1C, C12H1orf115, FSTL1, PTGFRN and IFIH1 in the gene modules connected with growth and slaughter performance. Our research identifies the hub genes associated with the growth and slaughter performance of Hu sheep, which play an important role in their muscle growth, organ and cartilage development, blood vessel development and energy metabolic pathways. Our findings might lead to the development of potentially-useful biomarkers for the selection of growth and slaughterer performance-related attributes of sheep and other livestock.

In this experiment, seven healthy male and seven female Hu sheep were selected under the same feeding conditions, and their meat quality and volatile metabolites were determined. The results showed that the longissimus dorsi muscle of ewe had better performance in tenderness and water retention (P<0.05). The content of water and crude protein in longissimus dorsi muscle of RAMS was higher (P<0.05), and the content of crude fat was lower (P<0.01). There was no significant difference in amino acid content. A total of 305 metabolites were detected in the samples of both sexes, mainly lipids, lipid compounds, sugars, carbohydrates and amino acids. Eleven key differential metabolites were obtained by analysis. Differential metabolites were enriched in 32 metabolic pathways. Among the 15 significantly enriched metabolic pathways, 10 were related to amino acid synthesis, and the metabolism of alanine, aspartate and glutamate had the greatest influence on the differential metabolites.

This study aimed to investigate the effects of various diluents on the quality of Hu ram sperm stored at 4 °C. Semen samples were collected from three Hu rams and diluted with diluents A (Sodium citrate-Glucose-Egg yolk), B (Sodium citrate-Glucose), C (Fructose-Skimmed milk powder-Soy lecithin), and D (Tris-Fructose-Citric acid-Egg yolk). Total motility (TM), straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), average motion degree (MAD), acrosome integrity, membrane integrity, and reactive oxygen species (ROS) were evaluated. The results showed that diluent D had better preservation in terms of the sperm TM, VSL, VCL, VAP, MAD, and membrane and acrosome integrity. On the third day of the storage, the sperm PM of diluent D was higher than that of other diluents (p < 0.05). The ROS level of diluent D was lower than that of other diluents on the fifth day (p < 0.05). On the seventh day of the storage, the sperm TM in diluent D reached 50%, which was the highest in all diluent groups. On the seventh day of the storage, the integrity of the sperm membrane and the integrity of the acrosome of the sperm in diluent D were the highest in all diluent groups (p < 0.05). In conclusion, these results indicated that diluent D improved the semen quality during storage at 4 °C. In this study, diluent D was the best diluent formula for Hu ram semen stored at 4 °C.