Sheep Sperm Research Articles

Environmental Heat Stress Decreases Sperm Motility by Disrupting the Diurnal Rhythms of Rumen Microbes and Metabolites in Hu Rams.

Heat stress (HS) has become a common stressor, owing to the increasing frequency of extreme high-temperature weather triggered by global warming, which has seriously affected the reproductive capacity of important livestock such as sheep. However, little is known about whether HS reduces sperm motility by inducing circadian rhythm disorders in rumen microorganisms and metabolites in sheep. In this study, the year-round reproduction of two-year-old Hu rams was selected, and the samples were collected in May and July 2022 at average environmental temperatures between 18.71 °C and 33.58 °C, respectively. The experiment revealed that the mean temperature-humidity index was 86.34 in July, indicating that Hu rams suffered from HS. Our research revealed that HS significantly decreased sperm motility in Hu rams. Microbiome analysis further revealed that HS reshaped the composition and circadian rhythm of rumen microorganisms, leading to the circadian disruption of microorganisms that drive cortisol and testosterone synthesis. Serum indicators further confirmed that HS significantly increased the concentrations of cortisol during the daytime and decreased the testosterone concentration at the highest body temperature. Untargeted metabolomics analysis revealed that the circadian rhythm of rumen fluid metabolites in the HS group was enriched by the cortisol and steroid synthesis pathways. Moreover, HS downregulated metabolites, such as kaempferol and L-tryptophan in rumen fluid and seminal plasma, which are associated with promotion of spermatogenesis and sperm motility; furthermore, these metabolites were found to be strongly positively correlated with Veillonellaceae_UCG_001. Overall, this study revealed the relationship between the HS-induced circadian rhythm disruption of rumen microorganisms and metabolites and sperm motility decline. Our findings provide a new perspective for further interventions in enhancing sheep sperm motility with regard to the circadian time scale.

Influence of liquid extender, preservation temperature and time on the sheep sperm quality.

In this study, we evaluated sheep sperm quality after using Tris-citrate-fructose-based extender with and without egg yolk, a Tris-citrate without fructose and with egg yolk and the commercial extender Biladyl®, preserving diluted semen at 15 and 23°C for different times (4, 24, 48 and 72 h). The results showed that the diluents with fructose and egg yolk gave the best results of seminal quality. Moreover, the production of ROS was higher for the temperature of 23°C compared to the temperature of 15°C (control). In addition, VCL and the percentage of spermatozoa with intact acrosome decreased with temperatures of 23°C. Finally, a drastic decrease in sperm quality was observed after 24 hours of preservation for most of the parameters evaluated.

The influence of the conditioned environment of sheep mesenchymal stromal cells on the qualitative parameters of sperm cells

The purpose of this research was to study the influence of the conditioned medium of mesenchymal stem cells from adipose tissue and bone marrow of sheep on the quality parameters of sperm during a threehour incubation. The sperm of sexually mature rams of the Romanov and Dorper breeds at the age of 1-2.5 years was taken and examined. A total of 10 samples were taken (n=10). Conditioned medium (CM) was collected after 72 h of cultivation in mattresses with 90-100% cell monolayer. Three groups of samples, 10 in each, were formed: control (spermatozoa 7x1010 cells/ml + PBS buffer 100 μl, pH – 7.4), first experimental (spermatozoa 7x1010 cells/ml + conditioned medium (CM) MSC BM 2x108 cells/ml, pH – 7.2), the second experimental one (spermatozoa 7x1010 cells/ml + conditioned medium (CM) MSCs in adipose tissue 2x108 cells/ml, pH – 7.2). Comparisons of values were made at each incubation stage (0, 1, 2 and 3 hours) within each group. It was found that the use of conditioned medium of mesenchymal stem cells from adipose tissue and bone marrow of sheep at a concentration of 2x108 cells/ml and spermatozoa at a concentration of 7x1010 cells/ml in terms of improving the quality indicators of germ cells. During a three-hour incubation of sheep sperm with conditioned medium of mesenchymal stem cells (MS), a decrease in the number of progressively moving sperm was observed. At the same time, a statistically significant difference was recorded in the first experimental group (CS MSC BM) on the 3rd hour of incubation: 1.9 times (p<0.01), as well as in the second experimental group (CS MSC VT) on the 2nd and 3rd hour of incubation compared with indicators of 0 hour of incubation: 1.4 times (p≤0.05) and 2.4 (p<0.01), respectively. A statistically significant decrease in the number of morphologically normal sperm was recorded during an hour-long incubation with conditioned medium of MSCs from sheep bone marrow and amounted to 43.90±2.22% (p=0.04).

Neonatal vitamin A supplementation improves sheep fertility potential.

This study aimed to explore the effects of neonatal vitamin A (VA) supplementation on testis development and spermatogenesis. A total of 32 newborn lambs were intramuscularly injected with corn oil (control group) or corn oil + 2500 IU/kg BW VA (VA group). They were slaughtered and sampled at 3 weeks and 8 months of age to analyze spermatogenesis, cell proliferation, hormone secretion, antioxidant status of the testis, and adult sheep sperm parameters. Compared with the control group, the expression of spermatogonial differentiation-related genes in VA group was up-regulated (P < 0.05). Testis weight, seminiferous tubule diameter, number of spermatogonium and spermatocyte, and sperm density increased significantly in VA group at 8 months of age (P < 0.05). Neonatal VA injection upregulated the expression of the cell proliferation marker PCNA and cell cycle-related genes in the testis (P < 0.05). VA increased the concentrations of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum and upregulated steroidogenesis-related genes in the testis (P < 0.05). The antioxidant levels in the VA group were maintained at high levels. The total antioxidant capacity (T-AOC), antioxidant enzyme content and antioxidant-related genes were increased in the testis (P < 0.05). Furthermore, neonatal VA injection activated retinoic acid (RA) signaling to maintain the blood-testosterone barrier (BTB) in the testis of 3-week-old sheep. AMP-activated protein kinase (AMPK) and protein kinase B (AKT) signaling were also modulated in the sheep testis (P < 0.05). Taken together, VA supplementation in newborn rams promotes testis development and spermatogenesis to improve fertility.

Roles of Y-27632 on sheep sperm metabolism.

To investigate the effect of Y-27632 on low-temperature metabolism of sheep sperm, different concentrations of Y-27632 were added to sheep semen at 4°C in this experiment to detect indicators such as sperm motility, plasma membrane, acrosome, antioxidant performance, mitochondrial membrane potential (MMP), and metabolomics. The results showed that the addition of 20µM Y-27632 significantly increased sperm motility, plasma membrane integrity rate, acrosome integrity rate, antioxidant capacity, MMP level, significantly increased sperm adenosine triphosphate (ATP) and total cholesterol content, and significantly reduced sperm Ca2+ content. In metabolomics analysis, compared with the control group, the 20µM Y-27632 group screened 20 differential metabolites, mainly involved in five metabolic pathways, with the most significant difference in Histidine metabolism (P = 0.001). The results confirmed that Y-27632 significantly improved the quality of sheep sperm preservation under low-temperature conditions.

Metabolomics Analysis of Sodium Salicylate Improving the Preservation Quality of Ram Sperm.

The aim of this study was to investigate the effects of sodium salicylate (SS) on the preservation and metabolic regulation of sheep sperm. Under 4 °C low-temperature conditions, SS (at 10 µM, 20 µM, 30 µM, and 50 µM) was added to the semen diluent to detect sperm motility, plasma membrane, and acrosome integrity. Based on the selected optimal concentration of SS (20 µM), the effects of 20 µM of SS on sperms' antioxidant capacity and mitochondrial membrane potential (MMP) were evaluated, and metabolomics analysis was conducted. The results showed that on the 20th day of low-temperature storage, the sperm motility of the 20 µM SS group was 62.80%, and the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher than those of the control group (p < 0.01). The content of Ca2+, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly lower than those of the control group (p < 0.01), and the total antioxidant capacity (T-AOC) was significantly higher than that of the control group (p < 0.05); mitochondrial activity and the total cholesterol (TC) content were significantly higher than those in the control group (p < 0.01). An ultrastructural examination showed that in the SS group, the sperm plasma membrane and acrosome were intact, the fibrous sheath and axoneme morphology of the outer dense fibers were normal, and the mitochondria were arranged neatly. In the control group, there was significant swelling of the sperm plasma membrane, rupture of the acrosome, and vacuolization of mitochondria. Using metabolomics analysis, 20 of the most significant differential metabolic markers were screened, mainly involving 6 metabolic pathways, with the amino acid biosynthesis pathway being the most abundant. In summary, 20 µM of SS significantly improved the preservation quality of sheep sperm under low-temperature conditions of 4 °C.

Comparative characteristics of protocols for cryopreservation of ram semen

Purpose: study and comparison of protocols for cryopreservation of sperm of stud rams.Every day the popularity of sheep farming in Russia is increasing, since sheep are unpretentious in maintenance and at the same time they are multiproduct animals, providing the population with the necessary raw materials (wool, felt) and food products (meat, milk). For this reason, artificial insemination is of fundamental importance in the development of this branch of livestock farming, an important part of which is the correct cryopreservation of ram sperm. There are quite a few advantages of using cryopreserved sperm. Cryopreservation of sheep sperm contributes to the spread of the use of artificial insemination in this branch of the livestock industry. Freezing technology largely ensures the preservation of genetic material and its transportation, eliminating geographical barriers in the use of artificial insemination, and allows increasing the load on the ram sire without risk to his reproductive health. However, during the process of cryopreservation, sheep sperm receives irreversible damage due to cold shock, osmotic stress and oxidative processes, which lead to a decrease in the ability to fertilize sperm. Thus, chromatin may be damaged, membrane permeability increased, hyperoxidation and the formation of reactive oxygen species are possible, which affects the ability to fertilize. As a result, frozen ram semen may have low fertility. For this reason, it is extremely important to select an up-to-date freezing and thawing protocol with maximum recovery of viable and functional sperm of a given animal species for successful further use in artificial insemination. Technological advances in various fields of biotechnology have led to improvements in protocols, methods and equipment used in the laboratory, which have a positive impact on the reliability, accuracy of the procedure. This article summarizes research on the effects of different cryopreservation procedures, cooling rates, cooling times and thawing protocols on ram semen quality. In addition, a correct protocol for cryopreservation of Romanov breed sperm was formed, based on the results of our own research.

QUALITY OF FROZEN SHEEP SPERM USING SOYBEAN LECITHIN EXTENDER

The study aimed to study the impact of optimum levels of soybean lecithin in Tris extenders on the quality of frozen sheep sperm. The observed variable was the quality of the post-freezing spermatozoa, which covers the progressive motility, viability, abnormality and integrity of the sperm membrane. The results of the study showed that the level of lecithin in soybeans had a real influence (P&lt;0,05) on all parameters of the quality of post-freezing sheep spermatozoa. The higher the level of soybean lecithin, the higher the percentage of progressive motility, viability, integrity of sperm membranes up to 3% soybean lecithin, then a decrease of 4% soybean lecithin and decreasing sperm abnormality percentages to 3% Soybeans Lecithin. The Average percentage of progressive motility (43.79%), viability (50.85%) and membrane integrity (51.65%) of post-freezing spermatozoa treated with 3% soya bean lecithin (L3) was significantly higher (P&lt;0.05), and sperm abnormalities (13.72%) were significantly lower (P &lt;0.05) than all treatments tried.&#x0D; &#x0D; Keywords: Membrane integrity, Motility, Frozen spermatozoa Quality, Soybean lecithin

Identification and functional analysis of differentially expressed proteins in high and low freezing tolerance sheep sperm

The purpose of this study was to identify proteins associated with differences in the freezing tolerance of sheep sperm and to analyze their functions. Qualified fresh semen from four breeds of rams, the Australian White, white-head Dorper, Black-head Dorper, and Hu sheep breeds, were selected for cryopreservation. The sperm freezing tolerance was investigated by evaluation of the overall vitality, progressive vitality, and rapidly advance vitality of the sperm. A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and the protein functions were analyzed. It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. These two breeds were used for the construction of a model based on differences in freezing tolerance and the identification of sperm proteins expressed differentially before freezing and after thawing. A total of 128 differentially expressed proteins (88 up-regulated and 40 down-regulated) were identified before freezing and after thawing in Hu sheep sperm (fresh/frozen Hu sheep sperm referred to as HL vs. HF), while 219 differentially expressed proteins (106 up-regulated and 113 down-regulated) were identified in white-head Dorper sheep (fresh/frozen white-head Dorper sheep sperm referred to as WL vs. WF). A comparison of these differentially expressed proteins showed that 57 proteins overlapped between the two breeds while 71 were only expressed in Hu sheep and 162 were only expressed in white-head Dorper sheep. Functional annotation and enrichment analyses of differentially expressed proteins down-regulated in Hu sheep involved in phosphorylation of phosphatidylinositol phosphate kinases, regulation of GTPase activity and glycolysis/gluconeogenesis signaling pathway. Up-regulated proteins of Hu sheep participated in oxidoreductase activity and oxidative phosphorylation process of sperm freezing. Furthermore, down-regulated in white-head Dorper sheep involved in the metabolic regulation of carbohydrate and nuclear sugar metabolism. Up-regulated proteins of white-head Dorper sheep involved in the ferroptosis and oxidative phosphorylation pathways. Collectively, These proteins were found to participate mainly in oxidative phosphorylation as well as phosphorylation and metabolic processes in the mitochondria to affect the freezing tolerance of sheep sperm.

Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep.

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

TEPUNG BEKICOT (Achatina fulica) SEBAGAI PAKAN TAMBAHAN UNTUK MENINGKATKAN KUALITAS SEMEN DOMBA (Ovis aries)

One of the effort to provide superior rams is to pay attention to the provision of feed that has good substances and is able to improve the quality of sperm. Based on studies, snail flour has a fairly high protein content and snails can be an alternative source of protein in additional feed. This study aims to improve the quality of sheep sperm using an additional feed of snail flour (Achatina fulica) which can increase the use value and economic value. Eight sheep used in this research were divided into one group as the control and two groups as the treatment. The control group was given standard sheep forage, while the treatment groups were given standard sheep forage added with flour snail of 5% and 10%. Sperm collection using castration method. Parameters observed were motility, viability, and concentration of sperms. The results of the study showed that the addition of snail flour as additional feed for sheep affected sperm quality with a significant increase in concentration parameters, while motility and viability did not show a significant increase

PLCζ can stably regulate Ca2+ fluctuations in early embryo

Phospholipase C zeta (PLCζ) is an important inducer of Ca2+ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca2+ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca2+ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca2+ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca2+ in early embryos at different stages, Ca2+ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca2+ fluctuations differed. PLCζ resulted in Ca2+ peaks that appeared at the cleavage and morula stages of early embryos, and Ca2+ returned to normal levels at the morula stage. However, the Ca2+ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca2+ fluctuations at different stages during early embryonic development.

Ruminant-specific genes identified using high-quality genome data and their roles in rumen evolution

Ruminants comprise a highly successful group of mammals with striking morphological innovations, including the presence of a rumen. Many studies have shown that species-specific or lineage-specific genes (referred to as new genes) play important roles in phenotypic evolution. In this study, we identified 1064 ruminant-specific genes based on the newly assembled high-quality genomes of representative members of two ruminant families and other publically available high-quality genomes. Ruminant-specific genes shared similar evolutionary and expression patterns with new genes found in other mammals, such as primates and rodents. Most new genes were derived from gene duplication and tended to be expressed in the testes or immune-related tissues, but were depleted in the adult brain. We also found that most genes expressed in the rumen were genes predating sheep–sperm whale split (referred to as old genes), but some new genes were also involved in the evolution of the rumen, and contributed more during rumen development than in the adult rumen. Notably, expression levels of members of the ruminant-specific PRD-SPRRII gene family, which are subject to positive selection, varied throughout rumen development and may thus play important roles in the development of the keratin-rich surface of the rumen. Overall, this study generated two novel ruminant genomes and also provided novel insights into the evolution of new mammalian organs.

Chlorogenic Acid Improves Quality of Chilled Ram Sperm by Mitigating Oxidative Stress.

Simple SummarySheep sperm is extremely sensitive to reactive oxygen species (ROS) and can produce a large amount of ROS during chilling storage, leading to a decline in semen quality. Adding antioxidants is an important method to improve semen quality. Chlorogenic acid (CGA) is a kind of plant extract with an antioxidant capacity, which can effectively eliminate free radicals and improve the antioxidant capacity of semen. However, its role in the chilling storage of Hu ram semen is not clear. Therefore, CGA with different concentrations was added to chilling storage extender to investigate its effect on chilled ram sperm. The results of this study revealed that CGA with proper concentration had a positive effect on chilled Hu ram sperm and 0.8 mg/mL CGA had the best effect.The purpose of this study was to investigate whether the addition of chlorogenic acid (CGA) to a sheep semen extender could improve the quality of chilled sheep sperm. Ejaculates (n = 80) were collected from five Hu rams with an artificial vagina. The ejaculates were mixed and divided into five equal parts, diluted with a CGA-free Tris–egg yolk extender (control), or supplemented with 0.2, 0.4, 0.8, and 1.2 mg/mL. The sperm kinematic parameters (viability, progressive motility), functional integrity of plasma membrane and acrosome, adenosine triphosphate (ATP) concentration and antioxidant parameters (Catalase (CAT), Superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC), ROS level and Malondialdehyde (MDA) content) were evaluated during storage of the semen. The results indicated that: PM, plasmatic membrane integrity and acrosomal integrity in 0.8 mg/mL CGA were higher (p < 0.05) from day 1 to 5. The ROS level in CGA groups was lower than the control (p < 0.05). CAT, SOD, ATP, and T-AOC were highest at 0.8 mg/mL concentration within 1 to 5 days. The above results indicated that the right concentration of CGA improved the quality of Hu ram sperm during chilling storage.

Coenzyme Q10 improves the quality of sheep sperm stored at room temperature by mitigating oxidative stress.

The purpose of this experiment was to explore whether coenzyme Q10 (CoQ10 ) improves the quality of sheep semen stored at room temperature by attenuating oxidative stress. Semen was diluted without (control group), and with antioxidants (5, 50, 250, and 500 μmol/L CoQ10 ). Sperm kinetic parameters and plasma membrane integrity were determined, and the reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), total antioxidant capacity (TAOC), catalase (CAT), and superoxide dismutase (SOD) activity were evaluated on the fifth day of semen preservation. The results showed that compared with the control group, the progressive motility in the 50 μmol/L group was higher (p < 0.05) within 2-5 days, and the plasma membrane integrity of sperm was higher in the 50 μmol/L group. The ROS content in the 5 and 50 μmol/L groups was reduced. The MDA level was reduced in the CoQ10 supplementation groups (p < 0.05). Additionally, the CAT, SOD, TAOC, ATP and MMP levels in the 50 μmol/L group were higher than those in the control group (p < 0.05). In conclusion, CoQ10 improved the quality of ram semen by alleviating oxidative stress, and 50 μmol/L CoQ10 was the optimum concentration.

Effect of Cystamine on Sperm and Antioxidant Parameters of Ram Semen Stored at 4 °C for 50 Hours.

Physical and chemical changes caused by oxidative stress in the spermatozoa membrane can reduce spermatozoa function and even lead to death. Cystamine (NH2-CH2-CH2-SH, β-mercaptoethylamine) is a natural substance that modulates the endocrine and metabolic status of animals. This substance has antioxidant and anti-apoptotic effects by inducing intracellular cysteine accumulation. Cystamine is used to treat many diseases despite its many side effects. Sheep semen is sensitive to the stressful condition of chilling storage, which restricts semen storage for artificial insemination in commercial herds. The effect of cystamine on spermatogenesis is not yet fully understood. The present study aimed to investigate the effect of cysteamine addition to the sheep sperm extender during cooling storage on semen quality parameters. Sperm samples were collected from six Edilbayevskaya rams (2 and 3 years old, 70-85 kg). The samples were diluted by extender and supplemented with different concentrations of cysteamine (0, 1, 2, 5, and 10 mM) and cooled to 4ºC for 50 h. Motility parameters, membrane integrity, viability, lipid peroxidation, and mitochondrial activity of cooled semen were evaluated at 0, 25, and 50 h of cooling storage. Although cysteamine failed to affect semen quality at start time (0 hrs), extender supplementation with cysteamine improved sperm total motility, progressive motility, and mitochondrial membrane potential during storage periods (P≤0.01). Moreover, using 1 and 2 mM cysteamine functionally and viably improved (P≤0.01) sperm membrane compared to other treatments. Antioxidant potential (AOP), lipid peroxidation (LPO), and total glutathione (tGSH) (except AOP at 50 h) were significantly different after semen storage at 4 °C. Therefore, levels of AOP and tGSH were significantly increased by using cysteamine. Cysteamine supplementation (1 and 2 mM cysteamine) leads to lower levels of LPO (p<0.01) at 0, 25, and 50 h. Therefore, finding and using the best concentrations of cysteamine in a cooling extender could be effective in saving sheep semen against damages of the cooling storage process.

Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep.

Simple SummaryHu sheep sperm is highly susceptible to ROS during storage at room temperature. It is very important to use an antioxidant to ameliorate oxidative damage. Tau is an important amino acid peptide antioxidant with a wide range of biological effects. It can effectively scavenge free radicals, regulate reproductive function, improve immunity, and enhance its antioxidant capacity. However, the effects of Tau in the preservation of Hu sheep semen at room temperature are unclear. Therefore, Tau was added to Hu sheep semen preserved at room temperature to explore its effect on semen. The results showed that adding an appropriate concentration of Tau had a positive effect on Hu sheep semen preserved at room temperature; in particular, 20 mM Tau performed best.The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

مطالعه تاثیر آنتی اکسیدانی عصاره هیدورالکلی درمنه (Artemisia incana) بر کیفیت اسپرم منجمد-یخ گشایی شده قوچ مغانی

Maintaining fertility in frozen sperm is very important. To investigate the effect of Artemisia (Artemisia incana) extract on sperm cryopservability of Moghani ram spermatozoa, different levels of the plant extract (0, 2, 4, 6, 8, 10 and 12 ml per deciliter) were added after extraction to diluted semen with an extender containing citrate buffer. After chilling, the semen was packaged in 0.5 ml straws and stored inside the liquid nitrogen until the evaluation of qualitative properties. This experiment was designed with 4 replications ­(with 10 straws in each replicate) per experimental group. Evaluation of sperm viability using eosin-nigrosine staining showed that adding 4 ml of Artemisia extract per deciliter to sheep sperm diluent had a positive effect on sperm viability. Similarly, the HOST test (hypoosmotic swelling test) to examine the integrity of the plasma membrane showed that the treatment had the highest membrane integrity (P<0.05). The highest and lowest Total Motility (TM) was observed in experimental groups 4 and 10 ml of extract in diluted semen, respectively (P<0.05). Examination of peroxidative damage by measuring malondialdehyde showed the lowest amount of malondialdehyde production was seen in the treatment with 12 mg of extract per deciliter diluent that was significantly different from other experimental groups (P<0.05). According to the results of the present experiment, it seems that the use of 4 ml Artemisia extract per deciliter of the diluent has positive effects on the quality of sperm after freezing.

The fertilizing capacity of sheep sperm

The article presents the results of studying the fertilizing ability of the sperm of ramsproducers of different breeds. It was found that highest fertility on average for all seasons take used sperm were characterized by sheep inseminated with semen of sheep of Stavropol breed, a minimum – with seed of North Caucasus mutton-wool breed.

Study on the region-specific expression of epididymis mRNA in the rams.

The epididymis is divided into three regions including the caput, corpus and cauda. Gene expression profiles in different regions indicate the different functions of epididymis which are crucial for sperm maturation. In this study, three one-year-old rams was used as the experimental animal. Transcriptome sequencing technology was used to sequence mRNA in the caput, corpus and cauda of the epididymis. Based on the spatiotemporal-specific expression pattern in the epididymis, the mRNA expression profiles of the three parts of the epididymis were analysed. Region-specifically expressed genes were analysed by GO and KEGG analyses to screen the key genes involved in sheep sperm maturation. We obtained 129, 54 and 99 specifically expressed genes in the caput, corpus and cauda, respectively. And twenty specific expressed genes related to sperm maturation were used to construct functional networks. The heatmap showed that 6 genes of LCN protein family were highly expressed in the head of epididymis of sheep. We infer that sperm maturation is gradual in the epididymis and that there are significant differences in epididymal gene expression patterns between different species. This provides a data resource for analysing the regulatory mechanism of epididymis genes related to sperm maturation in rams.