Capacitation Research Articles

Effect of melatonin on ram sperm capacitation induced by epidermal growth factor

Effect of melatonin on ram sperm capacitation induced by epidermal growth factor

Di-2-ethylhexyl phthalate (DEHP) exposure induces sperm quality and functional defects in mice

Di-2-ethylhexyl phthalate (DEHP) harms mammalian testis development, yet the specific mechanism of its effect on sperm quality and function is unclear. In this study, male mice were administrated DEHP (200 mg/kg/day) via intragastric (i.g.) injection for 35 days. The sperm quality and function of DEHP-exposed mice were evaluated. DEHP exposure reduced the relative testis weight and serum testosterone levels. In addition, sperm count and motility parameters decreased significantly, which led to reduced sperm fertility characterized by reduced acrosome reaction rate, sperm-egg binding capacity and blastocyte formation. DEHP exposure decreased anti-oxidant indicators and the expressions of Cat, Sod1, Prdx6 and Sirt1 in the testis. DEHP-exposure also resulted in decreased proliferating cell nuclear antigen (PCNA) expression in mice testis, as well as the dose-dependent inhibition of the proliferation of GC-1 and GC-2 cells. These phenotypes may be related to increased cell apoptosis characterized by BAX/BCL2 and P53 up-regulation. DEHP exposure resulted in the down-regulation of SIRT1 and p-AKT in mice testis and decreased levels of GC-1and GC-2 cells. DEHP co-incubation with sperm in vitro resulted in decreased tyrosine phosphorylation and progressive motility, as well as p-AKT expression in capacitated sperm. Differential sperm proteomics identified 495 differentially expressed proteins, including 257 proteins down-regulated in the DEHP-exposure group. Bioinformatics analysis showed that proteins involved in sperm-egg interaction and fertilization processes were significantly down-regulated. Pathway analysis demonstrated that the adhesion pathway was enriched in down-regulated proteins, while the pathway associated with ribosomes was enriched in up-regulated proteins. Conclusively, DEHP exposure impaired male fertility by affecting sperm quality and function, and a pathway mediating the DEHP-induced decline in sperm quality and function was identified. The study provides additional information for understanding the molecular mechanisms of DEHP exposure and its effects on male reproduction.

Lactoferrin affects in vitro and in vivo fertilization and implantationin rats.

Lactoferrin (LF) is present in the oviduct, reduces in vitro gamete interaction, and affects sperm capacitation parameters in humans. Our aim was to investigate LF actions on further stages of the reproductive process in the Wistar rat model. Motile sperm were obtained from cauda epididymis to assess LF binding by direct immunofluorescence and LF effect on acrosome reaction (AR) using a Coomassie blue staining. After ovarian hyperstimulation of female rats, oocytes were surgically recovered and coincubated with motile sperm and different doses of LF to estimate the in vitro fertilization (IVF) rate. To evaluate the LF effect on pregnancy and embryo implantation, female rats (80days old) were placed with males and received daily intraperitoneal injections of LF during one complete estrous cycle (pregnancy experiments) or during the first 8 gestational days (implantation experiments). The number of pregnant females and live born pups was recorded after labor. Moreover, the number of implantation sites was registered during the implantation period. LF was able to bind to the sperm head, midpiece, and tail. 10 and 100μg/ml LF stimulated the AR but reduced the IVF rate. The administration of 100 and 200mg/kg LF significantly decreased the number of implantation sites and the litter size, whereas 100mg/kg LF declined the pregnancy rate. The results suggest that LF might interfere with the reproductive process, possibly interfering with gamete interaction or inducing a premature AR; nevertheless, the mechanisms involved are yet to be elucidated.

Cancer-testis antigen lactate dehydrogenase C4 as a novel biomarker of male infertility and cancer.

A unique lactate dehydrogenase (LDH) isoenzyme designated as lactate dehydrogenase C4 (LDH-C4) is found in mammalian mature testis and spermatozoa. Thus far, LDH-C4 has been well studied with regard to its gene and amino acid sequences, structure, biological properties, and peptide synthesis. Accumulating evidence has shown that LDH-C4 is closely related to spermatic energy metabolism and plays a critical role in sperm motility, capacitation, and fertilization. Defects in the catalytic activity of LDH-C4 are key to pathophysiological abnormalities underlying infertility. LDH-C4 was originally thought to be present only in mature testis and spermatozoa; however, recent studies have implicated LDH-C4 as a cancer-testis antigen (CTA), owing to its aberrant transcription in a broad spectrum of human neoplasms. This review highlights the recent findings on LDH-C4 with particular emphasis on its role in male infertility and tumors.

Omics insights into spermatozoa activation induced by Fetal bovine serum in viviparous black rockfish (Sebastes schlegelii)

Black rockfish (Sebastes schlegelii) is an economically important marine species with the characteristics of viviparity. The spermatozoa were transferred into the ovary by mating and stored for several months until fertilization. Little is known about spermatozoa activation and its mechanism in black rockfish. In this study, the suitable medium for spermatozoa activation in vitro was explored, and the underlying mechanism was studied by omics analysis. Fetal bovine serum (FBS) could significantly enhance spermatozoa motility in vitro. Omics analysis showed 559 differentially expressed genes (DEGs) and 1311 differentially methylated genes (DMGs) were identified after FBS treatment. Transcriptome analysis revealed that FBS-induced spermatozoa motility activation is associated with spermatozoa capacitation regulated by the cAMP-SRC-PKA, cGMP-PKG and phospholipase D signaling pathway. Spermatozoa capacitation-related gene hsp90aa1 and chemotaxis-related gene cxcr4 were two of the important DMGs. Methylome analysis further revealed that FBS-induced epigenetic modifications are involved in spermatozoa capacitation and chemotaxis. 36 overlaps were identified between DMGs and DEGs, of which five genes were demonstrated to play a role in spermatozoa physiology, required for flagellum stability and spermatozoa motility. The results could provide new clues for understanding spermatozoa activation's molecular mechanism and help establish activation and/or immobilizing media for improving either artificial fertilization or cryopreservation in black rockfish.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p<.05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p<.05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p<.05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Cortisol modulates Ca2+ signaling and acrosome reaction in human sperm.

Stress and elevated cortisol levels have negative effects on fertility, although there is controversy about the effect of cortisol on human sperm. One study reported that hydrocortisone (HC), the synthetic form of cortisol, does not activate CatSper channel but is able to inhibit its activation by progesterone (Pg). However, subsequent reports showed that HC has an agonist effect on CatSper, producing intracellular Ca2+ ([Ca2+ ]i ) increases. These studies were performed using only electrophysiological techniques and fluorometric measurements of Ca2+ . Therefore, it is important to evaluate the effects of different HC concentrations on human sperm physiology using other approaches and techniques. To analyze the effects of different HC concentrations on human sperm physiology and to evaluate the association of perceived stress, anxiety and depression with acrosome reaction (AR), and semen quality. In capacitated human sperm, we tested the effects of HC on [Ca2+ ]i mobilization and AR. Furthermore, the perceived stress and symptoms of anxiety and depression were evaluated with standardized psychometric tests. HC induces a low increase in [Ca2+ ]i compared to Pg response while, at higher concentrations, HC inhibits the Pg-induced Ca2+ response. Also, low concentrations of HC induce AR and when co-incubating it with Pg, the percentages of AR are decreased. Finally, the anxiety symptoms are negatively correlated with Pg induced AR and semen quality. HC has an agonist effect on [Ca2+ ]i mobilization and AR. However, our data also suggest that HC may compete for the binding site of Pg. Additionally, the association between anxiety and decreased AR could be explained by high cortisol levels. Our data showed that HC interferes with the Pg action. Therefore, the negative correlation between anxiety symptoms and Pg-induced AR is due to the accompanying high cortisol levels of this condition and its competitive action with Pg.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences.

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

Identification and Validation of Yak (Bos grunniens) Frozen-Thawed Sperm Proteins Associated with Capacitation and the Acrosome Reaction.

To achieve fertilization, mammalian spermatozoa must undergo capacitation and the acrosome reaction (AR) within the female reproductive tract. However, the effects of cryopreservation on sperm maturation and fertilizing potential have yet to be established. To gain insight into changes in protein levels within sperm cells prepared for use in the context of fertilization, a comprehensive quantitative proteomic profiling approach was used to analyze frozen-thawed Ashidan yak spermatozoa under three sequential conditions: density gradient centrifugation-based purification, incubation in a capacitation medium, and treatment with the calcium ionophore A23187 to facilitate AR induction. In total, 3280 proteins were detected in these yak sperm samples, of which 3074 were quantified, with 68 and 32 being significantly altered following sperm capacitation and AR induction. Differentially abundant capacitation-related proteins were enriched in the metabolism and PPAR signaling pathways, while differentially abundant AR-related proteins were enriched in the AMPK signaling pathway. These data confirmed a role for superoxide dismutase 1 (SOD1) as a regulator of sperm capacitation while also offering indirect evidence that heat shock protein 90 alpha (HSP90AA1) regulates the AR. Together, these findings offer a means whereby sperm fertility-related marker proteins can be effectively identified. Data are available via Proteome Xchange with identifier PXD035038.

Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test.

The failures of binding to the oocyte zona pellucida are commonly attributed to defects in the sperm recognition, adhesion, and fusion molecules. SPAM1 (sperm adhesion molecule 1) is a hyaluronidase implicated in the dispersion of the cumulus-oocyte matrix. Therefore, the aim of this study was to characterize the SPAM1 distribution in the different physiological conditions of human sperm. Specifically, we evaluated the location of the SPAM1 protein in human sperm before capacitation, at one and four hours of capacitation and after hyaluronic acid (HA) selection test by fluorescence microscopy. Sperm bound to HA were considered mature and those that crossed it immature. Our results detected three SPAM1 fluorescent patterns: label throughout the head (P1), equatorial segment with acrosomal faith label (P2), and postacrosomal label (P3). The data obtained after recovering the mature sperm by the HA selection significantly (p < 0.05) highlighted the P1 in both capacitation times, being 79.74 and 81.48% after one hour and four hours, respectively. Thus, the HA test identified that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. Overall, our results provide novel insights into the physiological basis of sperm capacitation and could contribute to the improvement of selection techniques.

Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens).

Mammalian spermatozoa are not mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanism of protein phosphorylation during frozen-thawed sperm capacitation and AR has not been well studied. In this study, the phosphoproteomics approach was employed based on tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment). The identification of 1,377 proteins with 5,509 phosphorylation sites revealed changes in phosphorylation levels of sperm-specific proteins involved in regulation of spermatogenesis, sperm motility, energy metabolism, cilium movement, capacitation and AR. Some phosphorylated proteins, such as AKAP3, AKAP4, SPA17, PDMD11, CABYR, PRKAR1A, and PRKAR2A were found to regulate yak sperm capacitation and AR though the cAMP/PKA signaling pathway cascades. Notably, the phosphorylation level of SPA17 at Y156 increased in capacitated sperm, suggesting that it is also a novel functional protein besides AKAPs during sperm capacitation. Furthermore, the results of this study suggested that the phosphorylation of PRKAR1A and PRKAR2A, and the dephosphorylation of CABYR both play key regulatory role in yak sperm AR process. Protein-protein interaction analysis revealed that differentially phosphorylated proteins (AKAP3, AKAP4, FSIP2, PSMD11, CABYR, and TPPP2) related to capacitation and AR process played a key role in protein kinase A binding, sperm motility, reproductive process, cytoskeleton and sperm flagella function. Taken together, these data provide not only a solid foundation for further exploring phosphoproteome of sperm in yak, but an efficient way to identify sperm fertility-related marker phosphorylated proteins.

PDE5 inhibitors and male reproduction: Is there a place for PDE5 inhibitors in infertility clinics or andrology laboratories?

The objective of this review study is to evaluate the therapeutic role of PDE5 inhibitors (PDE5is) in the amelioration of oligoasthenospermia in infertile males. PDE5is have a beneficial influence on the secretory function of the Leydig and Sertoli cells, the biochemical environment within the seminiferous tubule, the contractility of the testicular tunica albuginea, and the prostatic secretory function. In several studies, the overall effect of sildenafil and vardenafil increased quantitative and qualitative sperm motility. Furthermore, some studies indicate that PDE5is influence positively the sperm capacity to undergo capacitation under biochemical conditions that are known to induce the sperm capacitation process. Additional research efforts are necessary in order to recommend unequivocally the usage of sildenafil, vardenafil, or avanafil for the alleviation of male infertility.

Reproductive toxicity of ritonavir in male: Insight into mouse sperm capacitation

Since COVID-19 began in 2019, therapeutic agents are being developed for its treatment. Among the numerous potential therapeutic agents, ritonavir (RTV), an anti-viral agent, has recently been identified as an important element of the COVID-19 treatment. Moreover, RTV has also been applied in the drug repurposing of cancer cells. However, previous studies have shown that RTV has toxic effects on various cell types. In addition, RTV regulates AKT phosphorylation within cancer cells, and AKT is known to control sperm functions (motility, capacitation, and so on). Although deleterious effects of RTV have been reported, it is not known whether RTV has male reproduction toxicity. Therefore, in this study, we aimed to investigate the effects of RTV on sperm function and male fertility. In the present study, sperm collected from the cauda epididymis of mice were incubated with various concentrations of RTV (0, 0.1, 1, 10, and 100 μM). The expression levels of AKT, phospho-AKT (Thr308 and Ser473), and phospho-tyrosine proteins, sperm motility, motion kinematics, capacitation status, and cell viability were assessed after capacitation. The results revealed that AKT phosphorylation at Thr308 and Ser473 was significantly increased, and the levels of tyrosine-phosphorylated proteins (at approximately 25 and 100 kDa) were significantly increased in a dose-dependent manner. In addition, RTV adversely affected sperm motility, motion kinematics, and cell viability. Taken together, RTV may have negative effects on sperm function through an abnormal increase in tyrosine phosphorylation and phospho-AKT levels. Therefore, individuals taking or prescribing RTV should be aware of its reproductive toxicity.

The impact of various antioxidant supplementation on ram's sperm quality, fertilization, and early embryo development, in vitro

The in vitro embryo production (IVEP) is very stressful for gametes. Gametes are subjected during in vitro manipulation to many different types of stress; oxidative stress is the most prominent one, which will cause damage or alter the genetic material of the sperm and reduce the quality of the oocytes, and has a crucial impact on the possibility of developing embryos even after implantation. This study aimed to determine the influence of antioxidants on the achievement of In vitro culture (IVC) and sperm's ability to adhere to and penetrate further into In vitro maturated oocytes. For this purpose, we have incubated ram sperm using four different treatments in terms of antioxidants: melatonin, cysteamine, vitamin C, and vitamin E. They were incubated by the standard methods of maturation and capacitation of sperm. The oocytes were fertilized by spermatozoa that had been capacitated with two groups of fertilization media, the first group containing melatonin and the second group containing cysteamine. Compared with other groups, sperms treated with melatonin demonstrated hyperactivity, and the fertilization rate was significantly increased. As for the IVF medium containing melatonin, it was superior to cysteamine in embryo development rates. In conclusion, melatonin could be a promising tool for improving sperm competence for fertilizing oocytes and embryo development in sheep.

G-Protein Coupled Receptors in Human Sperm: An In Silico Approach to Identify Potential Modulatory Targets.

G protein-coupled receptors (GPCRs) are involved in several physiological processes, and they represent the largest family of drug targets to date. However, the presence and function of these receptors are poorly described in human spermatozoa. Here, we aimed to identify and characterize the GPCRs present in human spermatozoa and perform an in silico analysis to understand their potential role in sperm functions. The human sperm proteome, including proteomic studies in which the criteria used for protein identification was set as <5% FDR and a minimum of 2 peptides match per protein, was crossed with the list of GPCRs retrieved from GLASS and GPCRdb databases. A total of 71 GPCRs were identified in human spermatozoa, of which 7 had selective expression in male tissues (epididymis, seminal vesicles, and testis), and 9 were associated with male infertility defects in mice. Additionally, ADRA2A, AGTR1, AGTR2, FZD3, and GLP1R were already associated with sperm-specific functions such as sperm capacitation, acrosome reaction, and motility, representing potential targets to modulate and improve sperm function. Finally, the protein-protein interaction network for the human sperm GPCRs revealed that 24 GPCRs interact with 49 proteins involved in crucial processes for sperm formation, maturation, and fertilization. This approach allowed the identification of 8 relevant GPCRs (ADGRE5, ADGRL2, GLP1R, AGTR2, CELSR2, FZD3, CELSR3, and GABBR1) present in human spermatozoa that can be the subject of further investigation to be used even as potential modulatory targets to treat male infertility or to develop new non-hormonal male contraceptives.

Embryo production by in vitro fertilization in wild ungulates: progress and perspectives – A Review

Abstract Wild ungulates are of fundamental importance for balancing ecosystems, as well as being the species of economic interest. Increasing concern over the accelerated population reduction of these species has resulted in the development of assisted reproduction techniques, such as in vitro fertilization (IVF), as a tool for conservation and multiplication. In the present scenario, IVF protocols were developed based on the methodologies used for domestic ungulates. Nevertheless, owing to the physiological and reproductive differences among the species, several factors associated with IVF and its relationship with the characteristics of the species of interest require clarification. In vitro conditions for the collection and selection of female and male gametes, oocyte maturation, sperm capacitation, co-incubation of gametes, and embryonic development can influence IVF results. Therefore, the present review considers the main advances in the methodologies already used for wild ungulates, emphasizing the strategies for improving the protocols to obtain better efficiency rates. Additionally, we discuss the conditions of each IVF stage, with emphasis on aspects related to in vitro manipulation and comparability with the protocols for domestic ungulates.

Analysis of reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin.

Cypermethrin contamination was a potential threat to soil organisms. In the present work, reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin was investigated. It was found that earthworms could absorb and accumulate residual cypermethrin in soil, and also earthworm activities helped accelerate the degradation of cypermethrin in soil. The accumulation of cypermethrin in earthworms induced sperm damage, and cypermethrin not only caused the imbalance of calcium homeostasis in earthworm sperm cells by inhibiting earthworm sperm Ca2+-ATP and Ca2+-Mg2+-ATP enzyme activities but also caused barriers in acrosome reaction. It also affected sperm energy supply of earthworms by inhibiting the activity of Na+-K+-ATPase and Mg2+-ATPase of earthworm sperm. Meanwhile, the inhibition of acrosome enzyme activity of earthworm sperm by cypermethrin led to hinder fertilization and reduced cocoon production of earthworms, and the damage of cypermethrin to sperm of earthworm was a significant cause of its reproductive toxicity. The results of the evaluation of IBR index showed that reproductive toxicity of cypermethrin to earthworms reduced with the increasing time. The decreased reproductive toxicity of cypermethrin to earthworms at the later stage of exposure (42-56 d) might be due to a combination of reduced absorption of cypermethrin in soil by earthworms, decreased accumulation of cypermethrin in the body, and improved sperm capacitation.

PROTEIN TYROSINE PHOSPHORYLATION MEASUREMENT THROUGH FLOW CYTOMETRY MAY BE A POTENTIAL NOVEL BIOMARKER OF HUMAN SPERMATOZOA CAPACITATION AND FUNCTIONAL STATUS: A PILOT STUDY

To determine if the measurement of tyrosine phosphorylation (pTyr) could be validated as a novel biomarker of sperm capacitation that reflects the capacitation and functional status of a semen sample. Do pTyr levels differ among fertile and sub-fertile men?

Regulatory effects of differential dietary energy levels on spermatogenesis and sperm motility of yellow-feathered breeder cocks.

The semen quality of breeder cocks profoundly impacted the numbers of matched layer hens and the economic benefits of the poultry industry. Adequacy and balance of poultry nutrition, especially the energy provision, critically modulated the reproductive potential of breeder cocks, however, the underlying mechanism was still unclear. For the purpose of this study, a total of 90 yellow-feathered 13-week-old roosters with the same age in days and similar body weight (1,437 ± 44.3 g) were selected and randomly divided into the low energy diet (LE), the moderate energy diet (ME), and the high energy diet (HE) treatments. The phenotypic parameters related to reproduction include semen quality, fertility, and hatchability, and the testis morphological parameters, including seminiferous epithelium length (SEL), seminiferous tubule perimeter (STP), seminiferous tubule area (STA), and Johnsen score, were measured to investigate the regulatory effects of different energy diets on reproductive performances. Furthermore, spermatogenesis and sperm motility-related genes, which included the sry-related high mobility group box (SOX) gene family and sperm-associated antigen (SPAG) gene family, and mitochondria apoptosis-related genes, such as Cyt-C, Bcl-2, and Bax, were measured to determine the underlying mechanism of energy on the reproductive performances. The The results showed that the gonadosomatic index and sperm motility in the ME treatment significantly increased compared with the LE treatment. Chickens in the ME treatment showed a preferable performance of testis development, especially a significant increment of SEL and Johnsen Score, compared with the LE and HE treatments. Finally, spermatogenesis-related genes, which included SPAG6, SPAG16, SOX5, SOX6, and SOX13, and apoptosis-related genes of mitochondria, such as the Cyt-C and Bcl-2, were significantly upregulated in the ME treatment. This study concluded that proper energy provision stimulated regular energy metabolism for spermatogenesis and sperm capacitation, which finally increased semen quality and reproductive performances of breeder cocks.

Cryosurvival of buffalo semen extended in tris-egg yolk supplemented with a combination of different concentrations of butylated hydroxytoluene and taurine

Cryopreservation is accompanied with much detrimental premature sperm capacitation, decreased livability and fertility consequences. Taurine is considered as an antioxidant ameliorating the semen value post freeze-thawing. The purpose of this investigation was to elucidate the impact of Tris-diluent enriched with a mixture of taurine and different concentrations of butylated hydroxytoluene on buffalo bull semen preservability. Semen samples were extended in a Tris extender. Variable concentrations of butylated hydroxytoluene (BHT) (0.5, 1.0 and 2.0 mM) were set in ethyl alcohol in prewarmed (37oC) test tubes. Then taurine (60 mM) was added into each tube. Extended semen samples were added into the test tubes and put at 37oC for 5 minutes to permit the permeation of BHT into the spermatozoa. The control test tubes were Tris extender with (zero BHT and zero taurine). The test tubes were exposed to the freezing protocol. Semen was assessed and conception rate was carried out. The sperm membrane integrity (HOST) was eminently higher in TTB1 and TTB2 if compared to the control post cooling (Table 1). The post thawing results (Table 2) revealed significant improvement in sperm motility in TTB2 if compared to the control and other concentrations. Alive sperm % was kept in all concentrations as the control. Sperm abnormalities were kept in TTB2 and TTB3 as the control. Sperm membrane integrity (HOST) was significantly improved in all significantly as compared to the control. The acrosomal integrity was kept in TTB1 and TTB2 concentrations the same as the control The conception percent (Table 3) was higher in TTB2. It could be concluded that, the post cooling results exhibited the best semen quality in TTB1and TTB2. The post -thawing results revealed that, most improved sperm parameters and conception rate were in TTB2.