Goat Spermatozoa Research Articles

The effect of mesenchymal stem cells from the adipose tissue and bone marrow of goats on the quality of their sperm cells

The purpose of this research is to study the influence of mesenchymal stem cells from adipose tissue and bone marrow of goats on the quality parameters of their sperm. A complex evaluation of sperm quality (volume, concentration, morphology, motility) of Saanen and Czech goats (n=10) at the age of 1-2.5 years was carried out according to generally accepted methods and protocols. Adipose tissue (AT) and bone marrow (BM) were obtained after the slaughter of goats (n=6) on a peasant farm in the Volkhov district of the Leningrad region. After processing the obtained samples, mesenchymal stem cells obtained from adipose tissue and bone marrow of goats were isolated, accumulated and cryopreserved. Three groups of samples of 10 each were formed: control (spermatozoa 7x1010 cells/ml + PBS buffer 100 μl, pH – 7.4), first experimental (spermatozoa 7x1010 cells/ml + MSCs 2x1010 cells/ml, pH – 7.2), the second was experimental (sperm 7x1010 cells/ml + BM MSCs 2x108 cells/ml, pH – 7.2) and a four-stage assessment of sperm quality was carried out: after incubation of sperm with mesenchymal stem cells at a temperature of 38℃ for 0, 1, 2 and 3 hours. Based on the results obtained, the working protocol for the use of mesenchymal stem cells from adipose tissue and bone marrow for the sperm of breeding goats is the concentration of MSCs - 2x108 cells/ml and the concentration of goat spermatozoa - 7x1010 cells/ml. The studies conducted have established the positive effect of mesenchymal stem cells from the adipose tissue and bone marrow of goats on the quality indicators of their sperm. At the same time, spermatozoa remain viable during three-hour incubation with a progression of movements equal to 51.5±8.46 - 51.8±5.2% and a number of morphologically normal sperm equal to 50.40±2.21 - 51.10±3, 14% (3 hours after incubation, MSCs from adipose tissue and bone marrow, respectively).

DNase I rescues goat sperm entrapped by neutrophil extracellular traps

Artificial insemination has been a predominant technique employed in goat husbandry for breeding purposes. Subsequent to artificial insemination, sperm can elicit inflammation in the reproductive tract, resulting in substantial the accumulation of neutrophils. Recognized as foreign entities, sperm may become entrapped within neutrophil extracellular traps (NETs) released by neutrophils, thereby exploiting their properties of pathogen elimination. Deoxyribonuclease I (DNase I), which is known for disintegrating NETs and causing loss of function, has been utilized to ameliorate liver and brain damage resulting from NETs, as well as to enhance sperm quality. This study investigated the mechanism of sperm-induced NETs and further explored the impact of DNase I on NETs. Sperm quality was evaluated using optical microscopy, while the structure of NETs was observed through immunofluorescence staining. The formation mechanism of NETs was examined using inhibitors and PicoGreen. The findings revealed that sperm induced the formation of NETs, a process regulated by glycolysis, NADPH oxidase, ERK1/2, and p38 signaling pathways. The composition of NETs encompassed DNA, citrullinated histone H3 (citH3), and elastase (NE). DNase I protects sperm by degrading NETs, thereby concurrently preserving the integrity of plasma membrane and motility of sperm. In summary, the release of sperm-induced NETs leads to its damage, but this detrimental effect is counteracted by DNase I through degradation of NETs. These observations provide novel insights into reproductive immunity in goats.

Impact of glucose and pyruvate on adenosine triphosphate production and sperm motility in goats.

This study evaluates goat sperm motility in response to metabolic substrates and various inhibitors, aiming to assess the relative contribution of glycolysis and mitochondrial oxidation for sperm movement and adenosine triphosphate (ATP) production. In the present study, two main metabolic substrates; 0 to 0.5 mM glucose and 0 to 30 mM pyruvate were used to evaluate their contribution to sperm movements of goats. Using a 3-chloro-1,2-propanediol (3-MCPD), a specific inhibitor for glycolysis, and carbonyl cyanide 3-chlorophenylhydrazone as an inhibitor for oxidative phosphorylation, cellular mechanisms into ATP-generating pathways in relation to sperm movements and ATP production were observed. Data were analysed using one-way analysis of variance for multiple comparisons. Sperm motility analysis showed that either glucose or pyruvate supported sperm movement during 0 to 30 min incubation. However, the supporting effects were abolished by the addition of a glycolysis inhibitor or mitochondrial uncoupler, concomitant with a significant decrease in ATP production. Although oxidative phosphorylation produces larger ATP concentrations than those from glycolysis, sperm progressivity in relation to these two metabolic pathways is comparable. Based on the present study, we suggest that goat sperm use glucose and pyruvate to generate cellular energy through glycolysis and mitochondrial respiration pathways to maintain sperm movement.

Proteomic Analysis of Frozen–Thawed Spermatozoa with Different Levels of Freezability in Dairy Goats

The results of artificial insemination (AI) are adversely affected by changes in sperm motility and function throughout the cryopreservation procedure. The proteome alterations of frozen–thawed spermatozoa with various levels of freezability in dairy goats, however, remain largely unknown. To discover differentially expressed proteins (DEPs) and their roles in dairy goat sperm with high or low freezability (HF or LF), we conducted 4D-DIA quantitative proteomics analysis, the results of which are presented in this work. Additionally, we explored the underlying processes that may lead to the variations in sperm freezing resistance. A total of 263 DEPs (Fold Change > 2.0, p-value < 0.05) were identified between the HF group and LF group in frozen–thawed dairy goat spermatozoa. In our Gene Ontology (GO) enrichment analysis, the DEPs were mostly associated with the regulation of biological processes, metabolic processes, and responses to stress and cellular component biogenesis. Our Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that the DEPs were predominantly engaged in oxidative phosphorylation, N-Glycan biosythesis, and cysteine and methionien metabolism. A protein–protein interaction (PPI) network analysis revealed 14 potential proteins (NUDFB8, SDHC, PDIA4, HSPB1, etc.) that might influence the freezability of dairy goat sperm. These findings shed light on the processes underlying alterations in the proteome and sperm freezability, aiding further research on sperm cryopreservation.

Transcriptome Analysis Reveals Spermatogenesis-Related CircRNAs and LncRNAs in Goat Spermatozoa.

Mammalian spermatozoa comprises both coding and non-coding RNAs, which are traditionally believed to be a residual of spermatogenesis. The differential expression level of spermatozoal RNAs is also observed between fertile and infertile human, thereby anticipated as potential molecular marker of male fertility. This study investigated the transcriptome profile of goat (Capra hircus) spermatozoa. The sperm transcriptome was analyzed by three different methods viz. RLM-RACE, long-read RNA sequencing (RNAseq) in Nanopore™ platform, and short-read RNAseq in Illumina™ platform. The Illumina™ sequencing discovered 16,604 transcripts with 357 mRNAs having FPKM (fragments per kilobase per million mapped reads) of more than five. The spermatozoal RNA suite included mRNA (94%), rRNA (3%), miscRNA (1%), circRNA (1%), miRNA (1%), etc. This study also predicted circRNAs (127), lncRNAs (655), and imprinted genes (160) that have potential role in male reproduction. The gene ontology analysis revealed the involvement of spermatozoal RNA in regulating male meiosis (TET3, STAT5B), capacitation (ACRBP, CATSPER4), sperm motility (GAS8, TEKT2), spermatogenesis (ADAMTS2, CREB3L4), etc. The spermatozoal RNA were also associated with different biological pathways viz. Wnt signaling pathway, cAMP signaling pathway, AMPK signaling pathway, and MAPK signaling pathways having potential role in spermatogenesis. Overall, this study enlightened the suite of spRNA transcripts in goat and their relevance in male fertility for diagnostic approach.

Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa

BackgroundReactive oxygen species (ROS) are strongly linked with oxidative stress (OS) generated during the process of sperm cryopreservation. Indeed, cellular damage from ROS has been implicated during sperm cryopreservation which causes deterioration in sperm quality and antioxidant nanoparticles (NPs) have been successful in preventing such damage. The interaction of NPs with sperm cells has been less frequently explored in farm animals. ObjectiveThe present study explored the effect of NP supplementation on sperm ultrastructure, potential interaction with sperm membrane (plasma and acrosome membrane), heat shock protein (HSP) gene expression levels and sperm quality in cryopreserved buck semen. Materials and methodsThirty-two (32) ejaculates were collected from four (4) adult male bucks and then diluted in Tris- citric acid- fructose- egg yolk (TCFY) extender containing the Zinc-oxide (ZnO) and Selenium (Se) NP treatments (T0: Control; TZn: 0.1 mg/mL ZnO NPs and TSe: 1 µg/mL Se NPs) after initial evaluation. Diluted semen was packed in 0.25 mL French mini straws and then stored in liquid nitrogen (LN2). Sperm parameters, lipid peroxidation (LPO) profile, sperm head morphology ultrastructural classification under transmission electron microscope (TEM), potential interaction of NPs with sperm membrane and expression of HSP genes were evaluated in the different treatment groups. ResultsWe found a significant (p < 0.05) increase in the percentage of spermatozoa with intact plasma membrane, and intact acrosome in the ZnO (0.1 mg/mL) and Se (1 µg/mL) NP supplemented groups in comparison to the frozen control group. TEM assessment revealed no internalization of both ZnO and Se NPs into the sperm structure. Few occasional contacts of ZnO NPs with the sperm membrane and a few agglomerates of Se NPs around the area of damaged membranes were visualized. HSP70 and HSP90 mRNA levels were significantly (p < 0.001) higher in the NP supplemented groups in comparison to the control. HSP70 and HSP90 mRNA levels had a strong positive association with sperm motility and a weak to moderate association with other sperm parameters. ConclusionsCurrent findings indicated that ZnO NPs are more potent than Se NPs in ameliorating peroxidative damages during sperm cryopreservation, increases semen quality parameters possibly by increasing the expression levels of HSP genes in buck semen. Furthermore, NP supplementation may have a potential role in preserving sperm head ultrastructure by acting as an antioxidant and reducing OS during various degrees of cellular insults, which needs to be further explored.

Combined addition of L-carnitine and L-proline improves cryopreservation of dairy goat semen

Cryopreservation of semen renders artificial insemination easier and cheaper compared to use of fresh semen. However, the cellular oxidative stress, toxicity of cryoprotectants, and osmotic imbalance may lead to a decline in semen quality and fertilization ability during the process of cryopreservation. L-carnitine and L-proline have been demonstrated to possess effective antioxidant properties in cryopreservation, with the latter also exhibiting excellent permeability and thus being utilized as a permeable cryoprotectant in the field. The aim of this study was to investigate the effects of LC and LP on cryopreservation of semen of dairy goats. After thawing, sperm motility, membrane integrity, and acrosome integrity rate of cryopreserved semen treated with LC (50 mM) were significantly higher compared to the untreated control samples. Based on this premise, we conducted experiments to assess the cryoprotective efficacy of different concentrations of LP. The findings demonstrated that the inclusion of 50 mM LP resulted in improved sperm motility compared to other concentrations. Furthermore, the levels of damaging reactive oxygen species and the malonyldialdehyde marker for oxidative stress were significantly lower in goat semen treated with these concentrations of LC and LP compared to semen exposed to other treatments. Semen treated with LC and LP also exhibited good fertilization ability during both in vitro fertilization and artificial insemination. Thus, LC (50 mM) and LP (50 mM) improve cryoprotection of dairy goat sperm which suggests that addition of these compounds will be highly beneficial to the development of dairy goat breeding.

Increase of the motility of buck semen with Andromed® extender and the addition of HTF

Objective: To test whether HTF (Human Tubal Fluid) is capable of protecting goat sperm cells increasing the semen m Objective: To determine if HTF (Human Tubal Fluid) can protect buck sperm cells, increasing semen motility when used by itself or combined with the Andromed® commercial extender. Design/Methodology/Approach: The semen of eight bucks of the Boer breed was used (10 ejaculates per male). Each ejaculate was diluted in three treatments (5 repetitions per treatment): 1) only Andromed®; 2) only HTF; and 3) Andromed® + HTF. The following variables were evaluated: mass motility (MM) and progressive motility (MP) in fresh (37 °C) and refrigerated (5 °C) semen. The differences between treatments were detected, based on the MM and MP in fresh and refrigerated semen variables, using the GLM procedure (SAS V9). In the case of the MM evaluated in three different periods, the measures repeated over time were analyzed using the Proc Mixed procedure (SAS V9). Results: Regarding the quality of fresh spermatozoa, MM and MP recorded similar values (p&gt;0.05) for the three treatments. On the one hand, MM registered 97.1% for Andromed®, 95.8% for HTF, and 97.5% for Andromed® + HTF; its highest value was achieved with the Andromed® + HTF combination, followed by Andromed® and HTF. On the other hand, the highest MP percentage was observed with the combination of Andromed® + HTF (91.0%), followed by Andromed® (90.4%), and HTF (89.0%). Regarding the quality of the sperm refrigerated at 5 °C, the combination of Andromed® + HTF had a higher MP value (p&lt;0.05) than other treatments. Study Limitations/Implications: In all the evaluated treatments, the motility of sperm decreased over time (0 to 24 hours after cooling); however, the treatments with Andromed®, and Andromed®+HTF maintained a high motility. Findings/Conclusions: The combination of Andromed® and HTF can be used to dilute both fresh and cooled semen.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

Effect of arginine-induced motility and capacitation on RNA population in goat spermatozoa.

In vitro capacitation is essential in assisted reproductive technologies (ART) for embryo production. Recently, arginine has been proven to enhance capacitation in mammalian spermatozoa. However, the detailed mechanism of action of arginine remains elusive. This study investigated the effect of arginine-induced capacitation and motility enhancement on the spermatozoal RNA (spRNA) population in goats. Goat spermatozoa were treated with arginine for up to six hours and compared with non-treated or PHE (penicillamine, hypotaurine, and epinephrine)-treated spermatozoa at different intervals (0, 1, 2, 4, and 6 hours). Sperm parameters, including viability, individual motility, capacitation, acrosome reaction, and ROS production, were evaluated. The spRNA population was analyzed by short-read RNA sequencing (RNA-seq). The percentage of capacitated (73.21 ± 4.22%) and acrosome reacted (18.35 ± 0.56%) spermatozoa was highest in arginine treatment, while PHE treatment showed the highest percentage (79.82 ± 4.31%) of motile spermatozoa from 0 to 4 hours of incubation. RNA-seq analysis identified 1,321 differentially expressed genes (DEGs) in arginine-treated spermatozoa compared to the control. The PGK2, RNASE10, ODF1, and ROPN1L genes involved in sperm motility and ACR, DKKL1, KCNJ11, and PRND genes involved in the capacitation process were upregulated in arginine-treated spermatozoa. The DEGs regulate sperm capacitation-related cAMP-PKA, PI3-Akt, calcium, and MAPK signaling pathways. The arginine-induced capacitation and enhanced sperm motility were associated with the upregulation of several genes involved in sperm motility and capacitation pathways. The comparative study also suggests that arginine may be used in lieu of PHE for motility enhancement and in vitro capacitation of goat spermatozoa.

Viability of Spermatozoa in Goat's Liquid Semen after Adding Sweet Orange Essential Oil to The Andromed Extender

Aims: To find out the percentage of live spermatozoa after diluting the goat's liquid semen using an andromed extender plus sweet orange essential oil.&#x0D; Study Design: Randomized Block Design.&#x0D; Place and Duration of Study: Sample: Galang, Deli Serdang Regency, Indonesia, between December 2022 and January 2023.&#x0D; Methodology: The research procedure starts with the preparation of semen extender, collection of fresh semen, dilution of semen and equilibration. This research obtained conducted using a Randomized Block Design consisting of 5 treatment levels and five replications. Semen storage using 3 Bucks, which has done for three days. As a treatment is the addition of sweet orange essential oil as much as (P0) 0%, (P1) 0.25%, (P2) 0.5%, (P3) 0.75% and (P4) 1% on the andromed extender. The observed variable was percentage viability spermatozoa evaluated before equilibration and after equilibration.&#x0D; Results: The results showed that the addition of sweet orange essential oil had a not significant effect (P &lt;0.01) to viability of spermatozoa. The results of adding sweet orange essential oil to the extender (after equilibration) were 80% (P0), 80% (P1), 80% (P2), 80% (P3) and 81% (P4).&#x0D; Conclusion: Adding sweet orange essential oil to andromed had no effect on the viability of goat spermatozoa in liquid semen.

Glucose Starvation Inhibits Ferroptosis by Activating the LKB1/AMPK Signaling Pathway and Promotes the High Speed Linear Motility of Dairy Goat Sperm.

In mammals, sperm acquire fertilization ability after capacitation in vitro or when in the female reproductive tract. The motility patterns of sperm undergo continuous changes from the moment of ejaculation until fertilization in the female reproductive tract. In vitro, hyperactivated motility can be induced through high glucose mediums, while in vivo, it is induced by oviduct fluids. Conversely, sperm maintain linear motility in seminal plasma or uterine fluids that contain low glucose levels. In dairy goat sperm, energy metabolism associated with capacitation depends on the energy sources in vitro, seminal plasma, or the female reproductive tract, especially the glucose levels. However, there is little experimental knowledge that glucose levels affect sperm energy metabolism in dairy goats. To clarify these hypotheses, we incubated dairy goat spermatozoa with different concentrations of rotenone-glucose (ROT), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and tigecycline (TIG) in vitro. Sperm motility attributes, ATP content, pyruvate and lactate levels, mitochondrial permeability transition pore fluorescence intensity, mitochondrial membrane potential (MMP), and protein synthesis were analyzed. Sperm motility patterns changed from circular to linear under low glucose conditions compared with those in high glucose conditions and showed a significant improvement in progressive motility and straight line speed, whereas lactate and pyruvate levels and MMP decreased remarkably. Incubation of spermatozoa with ROT, FCCP, and TIG inhibited sperm mitochondrial activity, protein synthesis, oxidative phosphorylation, and ATP levels, thereby reducing sperm motility, including the progressive motility, straight line speed, and total motility. Simultaneously, incubation of spermatozoa with Compound C under low glucose conditions significantly decreased the ATP levels and MMP, as well as liver kinase B1 and AMPK protein expression. Under low glucose conditions, sperm mainly produce ATP through mitochondrial OXPHOS to achieve high speed linear movement, inhibit ferroptosis through the LKB1/AMPK signaling pathway, and further maintain energy metabolism homeostasis.

Analyzing the effect of heparin on in vitro capacitation and spermatozoal RNA population in goats

Heparin is a glycosaminoglycan polymer that is commonly used as an anticoagulant. Heparin also induces in vitro capacitation in spermatozoa, although its molecular mechanism is elusive. This study investigated the effect of heparin on in vitro capacitation and spermatozoal RNA (spRNA) population in goats. Goat spermatozoa were treated with 20 μM heparin for 0–6 h and evaluated for motility, capacitation, acrosome reaction, and spRNA population by RNA sequencing (RNA-seq). It was observed that heparin enhanced sperm motility up to 6 h of incubation (p < 0.05). Heparin also induced capacitation and acrosome reaction within 4 h. RNA-seq identified 1254 differentially expressed genes (DEGs) between heparin-treated and control spermatozoa. Most DEGs (1251 nos.) were upregulated and included 1090 protein-coding genes. A few genes (PRND, ITPR1, LLCFC1, and CHRM2) showed >5-fold increased expression in heparin-treated spermatozoa compared to the control. The upregulated genes were found to be involved in cAMP-PKA, PI3-Akt, calcium, MAPK signaling, and oxidative stress pathways. DCFDA staining confirmed the increased oxidative stress in heparin-treated spermatozoa compared to the control (p < 0.05). In conclusion, the results of the present study suggest that heparin enhances sperm motility and induces capacitation by upregulation of the spRNA population and oxidative stress pathway.

Ethanolic Purslane (Portulaca oleracea) Leaf Extract Improves the Quality of Cryopreserved Goat Semen

Portulaca oleracea contains a wide range of chemical constituents, including flavonoids, alkaloids, polysaccharides, fatty acids, terpenoids, sterols, proteins, vitamins, and minerals. These chemical constituents are linked to the plant’s diverse pharmacological properties, such as antimicrobial and antioxidant activities. Therefore, this study is aimed at evaluating the effect of adding two concentrations (50 or 100 μg/mL) of Portulaca oleracea leaf ethanolic extracts in a native form (POLE) or nanoformulation (POLENF) on the antioxidant status of sperm medium to improve goat sperm freezability. Semen was evaluated postequilibration (5°C for 2 h), postthawing, and 2 h postincubation (37°C and 5% CO2). Phytochemical analysis showed the presence of 30 compounds in POLE, including phenolic acids and flavonoids and their glucosides. Total flavonoids were higher in POLE than in POLENF. Both extracts showed strong free radical scavenging capacity by the 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay, which was higher for POLENF than POLE, whereas the antioxidant activity by the ferric reducing antioxidant power assay had no differences. In comparison to free extender, the addition of POLE (100 μg) or POLENF (50 and 100 μg) significantly improved sperm motility and viability postequilibration and postincubation and antioxidant status of sperm medium postthawing. All POLE and POLENF concentrations significantly improved motility, viability, membrane integrity, and apoptosis of spermatozoa postthawing. Enzyme activities of AST, ALT, and LDH in sperm medium postthawing were not affected by treatments. In comparing different types and concentrations of POLE with free extender, the addition of POLENF (100 μg/mL) to goat semen extender gave the most valuable results as an effective free radical scavenger and antiapoptotic during cryopreservation.

Correlation Beetwen Body Weight with Scrotal Circumference, Testis Weight and Sperm Production of Boer Buck Intensively Rearing

Body weight of one of the regulators that determine the scrotum circumference and weight (large) testicles. Next, the weight of the testes has a very close relationship with sperm production that will determine the reproductive ability of a male. The purpose of this study was to determine the relationship between body weight and scrotum circumference, testicular weight and boer goat sperm production that was maintained intensively. The study was conducted with a descriptive method with the material of 15 male goats owned by PT. Sadhana Arif Nusa. Data is processed by regression and correlation analysis using the SPSS program. The results showed that the body weight average was 52.58 ± 14.96 kg, correlated very significantly (p &lt;0.01) with scrotum circumference (25.68 ± 1.96 cm) and testicular weight (0.46 ± 0, 10 kg) with a correlation coefficient of 0.67 and 0.91. Body weight not correlated (p&gt; 0.05) with good sperm production volume (0.50 ± 0.19 ml), pH (6.43 ± 0.19), sperm consistency, progressive motility (73.33 ± 7, 24 %) and Spermatozoa concentration (2.18 ± 0.54 x 109/ml). The scrotum circumference is very significantly correlated (p &lt;0.01) with a testicular weight and a real correlation (p &lt;0.05) with sperm volume, motility and concentration of spermatozoa with a coefficient of 0.91 each; 0.58; 0.56 and 0.54. The testes weight is real correlating (p &lt;0.05) with sperm volume, progressive motility and spermatozoa concentration with a coefficient of 0.53 each; 0.56 and 0.54. CONCLUSION, body weight correlates piosif with scrotum circumference, the weight of the quality of the testes and sperm production in boer goats.

Carboxylated ε-Poly-l-lysine Improves Post-Thaw Quality, Mitochondrial Functions and Antioxidant Defense of Goat Cryopreserved Sperm.

Carboxylated ε-poly-l-lysine (CPLL), a novel cryoprotectant, can protect the sperm membranes by inhibiting ice crystal formation during the cryopreservation process. The present study was conducted to investigate the consequence of CPLL supplementation on the post-thaw quality of cryopreserved goat sperm. For this, different doses (0, 0.5%, 1%, 1.5%, and 2%; v/v) of CPLL were added to the cryopreservation medium, and the motility, membrane and acrosome integrity, mitochondrial membrane potential (MMP), ATP level, ROS production, anti-oxidant defense system, malondialdehyde (MDA) level, and apoptosis in post-thaw sperm were evaluated. It was observed that the addition of 1% CPLL significantly (p < 0.05) increased the total motility, membrane integrity, acrosome integrity, and catalase (CAT) activity of post-thaw sperm compared to those of control and other CPLL doses. The ATP content was observed significantly (p < 0.05) higher in 0.5% and 1% CPLL, however, the SOD activity and progressive motility were significantly (p < 0.05) increased by adding CPLL at 1% and 1.5% level. Moreover, the addition of CPLL at 1% dose not only showed a lower percentage of apoptosis, but also significantly (p < 0.05) increased the MMP while reducing ROS production and MDA levels compared to those of other CPLL doses and/or control. Therefore, it is clear that the supplementation of 1% CPLL can remarkably improve the post-thaw goat sperm motility, membrane and acrosome integrity, antioxidant abundance, mitochondrial potentials, and ATP supply by protecting the sperm from cryodamage and undergoing apoptosis. These findings will provide novel insights into sperm cryobiology.

Post-Thaw Quality of Spermatozoa Frozen with Three Different Extenders in the Murciano Granadina Goat Breed.

Artificial insemination (AI) with frozen semen in goats still presents serious difficulties, especially in certain goat breeds, in spite of technological progress. The aim of this work is the in vitro study of seminal extenders adapted from those used on other species to evaluate the response of goat sperm to several homeostatic conditions in order to achieve optimal post-thaw semen quality. Three different extenders based on different activity principles were used: (1) extender according to the methodology proposed for pigs, (2) skimmed-milk-based extender according to the methodology proposed for goats in France, and (3) a new egg-yolk-based extender replacing membrane-protective surfactants with sodium dodecyl sulfate (SDS) and named by our team as extender "IMIDA". The freezing guidelines were those proposed for the freezing of porcine semen. The results obtained show that the egg-yolk-based extenders have good parameters of sperm motility at thawing, studied objectively using the computer-assisted sperm analysis (CASA) system and also subjectively. In particular, in the sperm resistance test after five hours, the thawed sperm containing SDS in their composition showed an optimal average on every evaluated parameter. The new IMIDA extender provided the highest sperm quality averages, so it could be a good extender to use in cryopreservation of semen in the caprine species.

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity.

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

In Vitro and In Vivo Studies on the Efficacy of Zinc-Oxide and Selenium Nanoparticle in Cryopreserved Goat (Capra hircus) Spermatozoa.

Different nanoparticles (NPs) are currently being investigated for their potential role as cryoprotectant during semen cryopreservation in several mammalian species. It may be possible to improve semen quality following cryopreservation by supplementation of NPs in the freezing extenders. The present study was carried out in semen collected from four (4) Assam Hill Goat bucks (10 ejaculates per buck) to investigate the effect of supplementing zinc oxide (ZnO) and selenium (Se) NPs in Tris-citric acid-fructose yolk (TCFY) extender on in vitro sperm quality and in vivo fertility rate after freeze-thawing. The size morphology and zeta potential of ZnO and Se NPs were evaluated prior to its incorporation in the freezing extender. Qualified semen samples (> 70% progressive motility) were divided into five (5) aliquots and then diluted in TCFY extender containing ZnO and Se NP supplementation at different concentrations (T0, control; T1, 0.1mg/mL ZnO NPs; T2, 0.5mg/mL ZnO NPs; T3, 0.5µg/mL Se NPs; and T4, 1µg/mL Se NPs). Diluted semen was packed in 0.25mL straws and then stored in liquid nitrogen. After thawing, post-thaw in vitro sperm attributes were evaluated. Finally, the effect of NPs on in vivo fertility rate was checked in heat-synched does (n = 70) by artificial insemination (AI) using straws that showed superior results during the in vitro study. Results showed that ZnO and Se NPs were poly-crystalline in nature with particle size below 100nm (nm). The evaluated post-thaw sperm in vitro attributes were significantly (p < 0.001) higher in T1 in comparison to T0. The antioxidant enzyme activities were significantly (p < 0.001) higher in T1. Lipid peroxidation (LPO) profile was significantly (p < 0.001) lower in T1. Sperm motility and mitochondrial membrane potential (MMP) had a highly significant (r = 0.580, p < 0.05) association in T1. No significant (p > 0.05) differences in pregnancy rates were recorded after AI in the different treatments. In conclusion, extender supplemented with 0.1mg/mL ZnO NPs improved post-thaw semen quality of goat spermatozoa consequently by increasing activities of endogenous antioxidant enzymes thereby lowering LPO levels. However, improved in vitro outcomes might not correspond to improved field fertility outcomes.

Immunocontraceptive Potential of Goat Zona pellucida as Monkey Population Control

A series of studies have explored the immunocontraceptive potential of zona pellucida-3 in local Indonesian goats. Goat zona pellucida-3 (gZP3) proteins have been identified and isolated. It has 82 kDa molecular weight and comprises 63.47% of the total goat zona pellucida protein. Immunofluorescence staining showed that gZP3 protein recognized goat sperm plasma membrane, whereas gZP3 antibodies recognized goat zona pellucida in native smear. In vitro fertilization study showed that gZP3 protein-supplemented in sperm capacitation medium and gZP3 antibody supplemented in oocyte maturation medium inhibited fertilization. It could be concluded that gZP3 was a fertilization receptor in a goat. Elisa and dot blot analysis showed that gZP3 protein was recognized by the serum of fertile unpregnant female mice. Therefore, it could be hypothesized that the gZP3 protein might be immunocontraceptive in mice. Immunization of mice with gZP3 proved that gZP3 prevented pregnancy and was dose-dependent. The contraceptive action of gZP3 was reversible after 14 estrous cycles. In vitro fertilization technique, it was proved that the antibody of gZP3 was able to prevent fertilization and decrease sperm binding rate on mice ova. The gZP3 nucleotide sequence has a homology at nearly 50% of the monkey ZP3 sequence (Macaca sp) but is foreign phylogenetically. The gZP3 protein has the potential to be developed as an immunocontraceptive substance for overpopulated monkeys to prevent agriculture disturbance in areas near forestry.