Ram Spermatozoa Research Articles

Analysis of the chemotactic response of ram spermatozoa to steroid hormones

Some substances in follicular fluid may be acting as sperm attractants, and steroid hormones progesterone (P4) and estradiol (E2), both secreted by the cumulus cells, are possible candidates. We have previously observed a chemotactic effect of the ewe follicular fluid in ram spermatozoa (data not published). The presence of progesterone and estrogen receptors in ram spermatozoa suggests the involvement of these hormones in the sperm functionality, including guided movement towards the oocyte. P4 acts as a chemoattractant in human and rabbit sperm, but there is no information on its effect, or E2 effect, on ram spermatozoa. The objective of this work was to study the putative chemotactic action of P4 and E2 on ram spermatozoa, analyzing the influence of the sperm capacitation state. Swim-up selected spermatozoa from nine Rasa Aragonesa rams were incubated without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail) to induce in vitro capacitation. After evaluating the sperm quality parameters in non- capacitated and incubated samples (motility, viability, capacitation state), IBIDI® µ-Slide Chemotaxis chamber (Ibidi GmbH) was loaded with swim-up, cap-TALP or cap-Cocktail sperm samples according to the manufacturer’s instructions. P4 or E2 were added to the chambers at 100 pM or 1 µM concentration each. Medium with 0.01% dimethyl sulfoxide (DMSO) was used as control treatment. After 5 min of equilibration, 200 fps videos were recorded for 3 sec with a video camera mounted on a microscope. Sperm trajectories and chemotaxis were analyzed with the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (versión 8.0.1). A chemotactic response was only observed in capacitated spermatozoa, as we previously described in presence of follicular fluid. P4 at both concentrations had a chemotactic effect on capacitated ram spermatozoa. However, E2 had no effect on any of the studied samples. These results reveal, for the first time, the chemotactic effect of P4 in ram spermatozoa. Grants: AGL-2017-83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).

Influence of the polymorphisms of the melatonin receptor 1A (MTNR1A) gene on the distribution of MT1 and MT2 melatonin receptors in ram sperm

Melatonin can exert some of its functions by binding to specific membrane receptors, mainly MT1 and MT2 in mammals. The melatonin receptor 1A (MTNR1A) gene exhibits several polymorphic sites. Certain RsaI genotypes have been related to an advance in the mating activity of ram-lambs or to a more intense reproductive behavior in adult rams. However, the influence of those genotypes in the location of melatonin receptors in ram spermatozoa remains unknown. The aim of this work was to evaluate whether RsaI polymorphisms of the MTNR1A gene influence the cellular distribution of MT1 and MT2 receptors in ram sperm. For this purpose, ejaculates from 18 previously genotyped Rasa Aragonesa rams were collected, and the location of melatonin receptors was assessed by indirect immunofluorescence, using anti-MT1 (Abnova Corporation, Cat# H00004543- A01, RRID: AB_462681) and anti-MT2 (OriGene, Cat#AP01322PU-N, RRID: AB_1619198) antibodies. Statistical analysis (χ²) was performed using SPSS Statistics v.26. For the MT1 receptor, G/A rams had a higher percentage of type I spermatozoa (labelling all over the head and tail) but lower rates of immunotypes II (reactivity at the equatorial and post-acrosomal regions, neck and tail) and III (only the equatorial region and tail labelled) when compared to G/G and A/A rams (P<0.01). Immunotype IV (only the flagelum stained) was also lower in G/A rams than in G/G (P<0.05). For the MT2 receptor, G/A rams had higher percentages of type AP spermatozoa (acrosome and post- acrosome equally stained), and lower percentages of type A (more intensity in acrosome than post-acrosome) when compared to G/G and A/A rams (P<0.001). Furthermore, G/G rams had a higher percentage of type P spermatozoa (more intense staining on post- acrosome than acrosome) than G/A and A/A rams (P<0.01). In conclusion, these results suggested that polymorphisms of the MTNR1A gene could influence the distribution of MT1 and MT2 receptors in ram sperm, which could have consequences on the direct effects of melatonin on spermatozoa. [Research supported by PID2020-113239RB-I00 and A07_20R. V.P-D. has a predoctoral contract from the Diputación General de Aragón]

Chemotaxis of capacitated ram spermatozoa to ewe follicular fluid

Sperm orientation mechanisms, as chemotaxis, are essential for the sperm to reach the oocyte and then fertilize it. The follicular fluid is the biochemical environment where the oocyte develops until its ovulation, and its composition changes throughout the estrous cycle. The follicular fluid is released together with the oocyte at the moment of ovulation, and it has been proposed as a chemoattractant for spermatozoa. This chemotactic ability has been demonstrated in some mammals, but to date, no studies have been done in the ovine species. Also, it has been reported that only capacitated and hyperactivated spermatozoa can respond to chemotactic stimuli. Therefore, the aim of this work was to study the chemotactic response of ovine spermatozoa to ewe follicular fluid, obtained at various stages of the estrous cycle, and to evaluate the influence of the capacitation status in the chemotaxis mechanism. In order to perform the experiments, ovine follicular fluids were obtained in the early and late follicular and luteal phases, and two concentrations, 2 and 10%, were tested. Semen samples were collected from nine Rasa Aragonesa rams, and spermatozoa were selected by the swim-up/dextran method. In vitro capacitation was induced by incubation without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail). After sperm quality evaluation (motility, viability, and capacitation state), the chemotactic assays were carried out with the aim of the commercial device (IBIDI® µ-Slide Chemotaxis chamber, Ibidi GmbH). Swim- up, cap-TALP or cap-Cocktail samples were loaded in the chambers in the presence of follicular fluid. Individual trajectories were analyzed by the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (Version 8.0.1). Follicular fluid at 2 and 10% concentrations, obtained in luteal and early follicular phases, induced sperm chemoattraction. This chemotactic response was only observed in capacitated spermatozoa, which supports previous evidence in other species. These results reveal, for the first time, the chemotactic effect of ewe follicular fluid in ram spermatozoa. Grants: AGL-2017- 83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).

Гематологічні показники та якість сперми баранів у період статевого спокою за згодовування ліпосомальної вітамінно-мінеральної добавки

The aim of the work was to investigate the effect of feeding a liposomal vitamin and mineral supplement during the period of sexual rest on hematological indicators and quality of ram sperm. The experiment was conducted at the PE “Kogut BM” in Horodotsky district, Lviv region, on 12 clinically healthy Texel rams, aged 2–4 years, during the period of sexual rest (March-May). Animals were divided into two groups — control and experimental, 6 animals per group. The control rams received the basic diet containing hay, corn silage, and compound feed. For 45 days, the sheep of the experimental group were individually added to the combined feed a feed additive containing vitamins A, D3, E, C and zinc gluconate in the form of a liposomal emulsion. At the beginning and at the end of feeding, we took blood samples, in which hematological indicators were determined. After the end of supplement feeding, ejaculates were collected twice a week in doublets for three weeks. Physiological indicators of ejaculate quality (volume, sperm concentration, percentage of live sperm, as well as sperm viability, morphological disorders and percentage of degenerative sperm) were determined using the CASA computerized system. It was established that feeding liposomal vitamin-mineral supplements to rams during the period of sexual rest improves hematological indicators: the content of erythrocytes, hemoglobin, platelets and hematocrit increase significantly (P&lt;0.05–0.001) while the content of leukocytes decreases by 24.2% (P&lt;0.05). Accordingly, the erythrocyte indices of the blood of experimental breeding rams were higher than those of control animals. Feeding rams with a liposomal vitamin and mineral supplement ensured an increase in ejaculate volume by 17.6% (P&lt;0.05), sperm concentration (P&lt;0.01), their viability, as well as a decrease in the number of immature (P&lt;0.001) and degenerated (P&lt;0.05) sperm. Higher quality indicators of ejaculates of rams under the influence of vitamins A, D3, E, C and zinc gluconate indicate the possibility of obtaining sperm from breeding rams during the period of sexual rest.

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

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

Topical reinforcement of the cervical mucus barrier to sperm.

Close to half of the world's pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives.

Motility Assessment of Ram Spermatozoa.

For successful fertilisation to occur, spermatozoa need to successfully migrate through the female reproductive tract and penetrate the oocyte. Predictably, poor sperm motility has been associated with low rates of fertilisation in many mammalian species, including the ram. As such, motility is one of the most important parameters used for in vitro evaluation of ram sperm quality and function. This review aims to outline the mechanical and energetic processes which underpin sperm motility, describe changes in motility which occur as a result of differences in sperm structure and the surrounding microenvironment, and assess the effectiveness of the various methods used to assess sperm motility in rams. Methods of subjective motility estimation are convenient, inexpensive methods widely used in the livestock industries, however, the subjective nature of these methods can make them unreliable. Computer-assisted sperm analysis (CASA) technology accurately and objectively measures sperm motility via two-dimensional tracing of sperm head motion, making it a popular method for sperm quality assurance in domesticated animal production laboratories. Newly developed methods of motility assessment including flagellar tracing, three-dimensional sperm tracing, in vivo motility assessment, and molecular assays which quantify motility-associated biomarkers, enable analysis of a new range of sperm motion parameters with the potential to reveal new mechanistic insights and improve ram semen assessment. Experimental application of these technologies is required to fully understand their potential to improve semen quality assessment and prediction of reproductive success in ovine artificial breeding programs.

Determination of Ram (Ovis aries) Sperm DNA Damage Due to Oxidative Stress: 8-OHdG Immunodetection Assay vs. SCSA®.

Conventional DNA analysis techniques can hardly detect DNA damage in ruminant spermatozoa due to high DNA compaction in these cells. Furthermore, these techniques cannot discriminate whether the damage is due to oxidative stress. The main purpose of this study was to evaluate the efficacy of two techniques for determining DNA damage in ovine sperm when the source of that damage is oxidative stress. Semen samples from twenty Manchega rams (Ovis aries) were collected and cryopreserved. After thawing, the samples were subjected to different levels of oxidative stress, and DNA oxidation was quantified using an 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunodetection assay and Sperm Chromatin Structure Assay (SCSA®). For this purpose, we evaluated five different concentrations of an oxidation solution (H2O2/FeSO4•7H2O) on ram sperm DNA. Our study with the 8-OHdG immunodetection assay shows that there are higher values for DNA oxidation in samples that were subjected to the highest oxidative stress (8 M H2O2/800 µM FeSO4•7H2O) and those that were not exposed to high oxidative stress, but these differences were not significant (p ≥ 0.05). The two SCSA® parameters considered, DNA fragmentation index (DFI %) and high DNA stainability (HDS %), showed significant differences between samples that were subjected to high concentrations of the oxidation agent and those that were not (p < 0.05). We can conclude that the 8-OHdG immunodetection assay and SCSA® detect DNA damage caused by oxidative stress in ovine sperm under high oxidative conditions; SCSA® is a more straightforward method with more accurate results. For these reasons, an oxidative-stress-specific assay such as 8-OHdG immunodetection is not needed to measure DNA damage caused by oxidative stress in ram sperm samples.

Alternative Opportunities to Collect Semen and Sperm Cells for Ex Situ In Vitro Gene Conservation in Sheep

The semen of domestic mammals is conventionally collected with an artificial vagina (AV) for artificial insemination (AI) or for short- or long-term storage. However, the procedure has certain drawbacks: animal training is not feasible in extensive animal care systems nor among wild species, as the trained animals sometimes fail to mount. Hence, there is a need for alternative semen collection methods. Electroejaculation (EEJ) and epididymal sperm recovery (ESR) are the two effective alternatives to AV. However, in recent years, animal welfare campaigners have called for the ban, in certain EU countries, of EEJ due to its inhumane nature. In this review, alternative methods of sperm collection (by EEJ and ESR, their qualities, and their freezing techniques) are highlighted, as well as the effects of EEJ on pre-freeze and post-thaw ram sperm quality parameters and the animal welfare progress made in EEJ between the 20th and 21st centuries. Additionally, the techniques for enhancing post-thaw sperm quality prior to freezing and for the freezing of EEJ and ESR spermatozoa are explored. ESR and EEJ are reliable alternatives to AV on certain occasions. EEJ is ideal for semen collection in wild or untrained animals, breeding soundness examinations, collection outside of the breeding season, and culling. At the same time, ESR is ideal in cases of castration, accidental death of elite sire, or postmortem for gene conservation purposes or assisted reproductive technologies (ARTs) studies.

Comparison of different poultry egg yolks-citrate extender with green tea (Camellia sinensis) extract addition on Sapudi ram spermatozoa quality in chilled temperature storage

Preservation of ram semen has an impact on the exposure of artificial conditions to the spermatozoa which was followed by overproduction of reactive oxygen species (ROS) and resulted in functional damages of spermatozoa. This study aimed to determine the effect of the combination of chicken, quail, and duck egg yolks with green tea extract (GTE) on the quality of Sapudi ram spermatozoa at 5°C storage. Ejaculates of Sapudi ram with more than 70% spermatozoa motility were divided into four groups. Semen were extended 1 : 10 in citrated-chicken egg yolk extender (control group, T0), and citrated yolk of chicken (T1), quail (T2), and duck (T3) egg added with 0.05 mg GTE per 100 mL of extender. Extended semen was kept in a tube, which was put in a beaker containing distilled water and stored in a refrigerator (5°C). Spermatozoa motility, viability, and plasma membrane integrity (PMI) were evaluated daily for five days. The results showed that GTE in duck egg yolk citrate extender could maintain the highest spermatozoa motility, viability, and PMI for five days (p &lt;0.05). It could be concluded that the duck egg yolk citrate extender with the addition of GTE (Camellia sinensis) was beneficial for maintaining the semen of Sapudi ram spermatozoa at chilled temperatures. Further study is needed to use the extender for freezing Sapudi ram semen.

Effects of Cell Concentration During Cryopreservation On the Post-thaw Quality of Santa Inês Ram Sperm

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe's cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G – 400, G – 800, G – 1200, and G – 1600 x 10 6 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 ºC, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 ºC/20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G – 400 treatment resulted in samples with the highest linearity and progressive motion (P &lt; 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P &lt; 0.05). Overall, use of the G – 400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G – 800 and G – 1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single rtificial insemination protocol is desirable.

The impact of varying doses of moringa leaf methanolic extract supplementation in the cryopreservation media on sperm quality, oxidants, and antioxidant capacity of frozen-thawed ram sperm

To increase rams’ post-thaw semen quality following cryopreservation, this study used enriched Tris-based diluent with varying amounts of moringa leaf methanolic extract (MLME). The antioxidant activity, total phenolic, and total flavonoid content were all assessed in MLME. The sperm of five healthy Awassi rams were collected, divided into 4 equal aliquots, and diluted [1:5; (v/v)] in Tris-citrate-glucose extender supplemented with 0.48, 0.56, and 0.64 mg MLME/ml or without MLME supplementation (control). The percentages of sperm total motility (STM, %), sperm progressive motility (SPM, %) and viability (V, %), abnormal morphology (AM, %), membrane functional integrity (MFI, %), and acrosome integrity (AI %) were measured. Malondialdehyde (MDA), nitric oxide (NO), ascorbic acid (AA), superoxide dismutase (SOD), glutathione peroxidase (GPx), total cholesterol (TC), low-density lipoproteins (LDL), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), zinc (Zn), and copper (Cu) were measured. The total phenolic gallic acid and flavonoid catechin (equivalent) contents were 19.78 mg/g and 11.94 mg/g, respectively. 2,2-Diphenyl-1-picrylhydrazyl (34.37 mM TE/g) and 2,2′-azino-bis/3-ethylbenzothiazoline-6-sulfonic acid (53.47 mM TE/g) were found in MLME. MLME had a 64.59 mM TE/g ferric-reducing power. In comparison to control, the addition of 0.64 mg/ml MLME to Tris-based extender resulted in the highest (P < 0.001) STM (55.22 ± 0.98), SPM (45.41 ± .70), SV (60.01 ± 1.05), MFI (75.23 ± 0.77), and AI (73.13 ± 0.72) and the lowest (P < 0.001) AM (21.34 ± 0.72) values. In comparison to the control, the addition of 0.56 mg/ml semen extender resulted in lower STM, SPM, SV, MFI, and AI with higher AM percentages. MDA (P = 0.03), NO (P = 0.012), CHO (P = 0.0001), and LDL (P = 0.004) were reduced by 0.64 mg/ml MLME, while AA (P = 0.017) and SOD (P = 0.0001) were elevated. In conclusion, the highest copper (P = 0.006) and lowest zinc concentrations in MLME (0.48 mg/ml extender) deteriorated the post-thaw semen quality, prompting us to suggest the addition of 0.64 mg MLME to rams’ Tris-based semen extender.

Vitamin E Lipid-Based Nanodevices as a Tool for Ovine Sperm Protection against Oxidative Stress: Impact on Sperm Motility.

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram (Ovis aries) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe2+/ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®®) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey’s post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

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.

Novelties in Ovine Assisted Reproductive Technologies – A Review

Abstract Artificial insemination (AI) as a part of assisted reproductive technologies represents the oldest and most widespread method used to accelerate genetic progress in all domestic animals. After its first implementation in ovine reproduction and almost 80 years afterward, AI is continuously used for improving the genetic merit, utilizing either fresh or short-time chilled semen. Nevertheless, regardless of the semen used for insemination, the conception rate (CR) is still lower in comparison to natural service. At least two factors are commonly thought to limit the success of the AI and reduce the CR: (1) failure of placing the semen directly into the uterus due to the specific anatomic structure of the ewe’s cervix; (2) lower viability of ram spermatozoa during cryopreservation (&lt;30% progressively motile spermatozoa after thawing). This review elaborates on recent studies that aimed to achieve acceptable CR through the implementation of cervical or intrauterine insemination: deep intracervical, intrauterine trans-cervical, and intracornual. Several hormonal treatments (oxytocin, estrogen, or prostaglandin) were evaluated on inducing cervical dilation that facilitates insemination. A comprehensive analysis was given to the effects of several antioxidants (GSSG, GSH, and cysteine) supplemented in ram semen-freezing media. Sex-sorted ram semen fertility rate results were presented from our studies.

Cooling storage of ram sperm in presence of antioxidant glutathione

In the process of cryopreservation, the semen of farm animals can be threatened by many factors, such as oxidative damage, which affect the motility and plasma membrane function of the sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of thawed sperm when added to the diluent of the semen. This study was aimed at examining the effect of GSH (given at 0.5 mM, 1.5 mM and 5 mM) on the motility and viability of ram sperm following storage under cooling conditions for several days. GSH reduced the negative effect of the cooling conditions on the total and progressive motility (CASA), stability of the membranes (annexin V/PI) and incidence of the apoptosis/necrosis (Yo-Pro-1/PI) in the sperm maintained at 5 °C for 24 h and 48 hours. However, this effect was neither reflected in the penetrating nor in the fertilising ability (zygotic cleavage rate) of the sperm examined in a heterologous (bovine oocytes and ram sperm) in vitro fertilisation test with GSH applied at 1.5 mM for 48 h of cooling storage. These results indicate that the addition of GSH to the sperm extender can maintain the quality of the ram sperm stored at cooling conditions for at least two days. To make a definite conclusion about the GSH effect on the sperm functionality (fertilising ability), other GSH concentrations should be tested.

Presence of leptin and its receptor in the ram reproductive system and in vitro effect of leptin on sperm quality.

Leptin is a 16 kDa hormone encoded by obese (OB) gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of mammals. Leptin and its receptor (OBR) have been found in male reproductive systems of human, bovine, equine and pig. The effects of leptin on sperm quality vary widely from different research findings. However, the presence of leptin and its receptor in the ram reproductive system and the in vitro effect of leptin on sperm quality have not reported yet. In the present study, we found that the OB was highly expressed in primary and secondary spermatocytes of the testes, OBR was highly expressed in secondary spermatocytes of the testes. The expressions of OB were in stereocilia of epididymis and in columnar cells of epididymal caput and cauda, the expressions of OBR were in columnar cells of epididymis and in stereocilia of epididymal corpus and cauda. The presence of both OB and OBR in testes, epididymis and sperm were confirmed through RT-PCR, immunolocalization and Western blot analyses. The RT-qPCR results indicated OB and OBR had higher expression levels in epididymal sperm than that of the ejaculated sperm in rams. When sperm were treated with 5 ng/mL leptin, the progressive motility (P < 0.01), straight-line velocity (VSL) (P < 0.05), average path velocity (VAP) (P < 0.05), membrane mitochondrial potential (MMP) (P < 0.01) and viability (P < 0.05) significantly increased, while DNA fragmentation index (DFI) and reactive oxygen species (ROS) significantly decreased compared to the control (P < 0.01), and the other semen parameters such as acrosome integrity and acrosome reaction rate had no significant changes between groups (P > 0.05). In conclusion, this is probably the first report describing localization of leptin and its receptors in the reproductive system of rams and their effects on sperm quality parameters. Our findings suggest that 5 ng/mL leptin treatment enhanced sperm motility, viability and MMP, and decrease DFI and ROS without obvious influence on the acrosome reaction in ram sperm. The potential mechanisms may be related to leptin’s ability to reduce the oxidative stress and apoptosis of sperms and improve their mitochondrial function and energy supply, therefore, to maintain the physiological homeostasis of the sperm.

Evaluation of Chicken Egg Yolk Plasma and Low-Density Lipoprotein Alone or Enriched with Ewe or Cow Skim Milk in Tris-Citric Acid-Based Diluent for Cryostorage of Ram Semen.

The main purpose of the current study was to find suitable and optimum levels of protectants among chicken egg yolk plasma (CEYP) and low-density lipoproteins (LDLs), alone or supplemented with ewe or cow skim milk, for cryopreservation of ram semen. In Experiments 1 and 2, the CEYP (28%) freezing extender was enriched with ewe or cow milk (2.5%, 5%, 10%, or 20%; v/v), respectively. In Experiments 3 and 4, the semen extender was prepared by varying the amounts of fresh or lyophilized LDL (lyo-LDL), respectively. Finally, ewe or cow skim milk was added to the freshly extracted LDL extender and the quality of frozen-thawed semen was examined (Experiments 5 and 6). Kinematics of spermatozoa (assessed using a computer-assisted sperm analysis system), viability, functionality of the plasma membrane, and levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated. Results revealed that addition of ewe or cow skim milk (5%, 10%, or 20%; v/v) to the CEYP diluent enhanced kinematics, viability, and membrane integrity of spermatozoa compared with the control (p < 0.05). Moreover, fresh LDL diluent was more effective than lyo-LDL in the cryosurvival of ram spermatozoa. In addition, enrichment of fresh LDL diluent with ewe or cow skim milk improved different variables of spermatozoa compared with the control (p < 0.05). Levels of MDA and TAC were not affected by adding ewe or cow milk to the diluents (p > 0.05). In conclusion, enrichment of fresh LDL extenders with ewe or cow milk also is proposed as an approach to preserve ram semen quality against cold shock and cryodamage injuries.

The Massive Impact of Ram's Sperm Starvation on the Fertilization and Blastocyst Rates in Terms of Sperm Quality and Capacitation

To obtain a high in vitro fertilization rate while ensuring the ideal development of the&#x0D; embryos, thus eliminating the most critical problems of assisted reproductive&#x0D; technology (ART) (implantation and low pregnancy rates). Therefore, sperm taken&#x0D; from the caudal epididymis must undergo the acquisition of progressive motility and&#x0D; fertilization. This process takes place through a series of physiological and biochemical&#x0D; changes, known as capacitation, a prerequisite for fertilization. In this work, we have&#x0D; developed a new way to incubate sperm using four different treatments in terms of&#x0D; energy substrates and different incubation methods. In this sperm energy restriction and&#x0D; recovery (RSE) treatment, after starving sperm for 50 min until they lost their capacity&#x0D; for motility, they were given glucose and incubated for 60 min. As for the other&#x0D; treatments, the sperm were not exposed to starvation and were incubated by the&#x0D; standard methods of maturation and capacitation of sperm, but with different energy&#x0D; substrates. According to the results obtained, the sperm was hyperactive and showed a&#x0D; significant increase in the ability to fertilize oocytes in the treatment that involved&#x0D; starvation and then saving the sperm by adding glucose, compared to other treatments.&#x0D; In conclusion, the effects of this feature persisted until after fertilization, which led to&#x0D; the production of a high percentage of blastocysts, as well as it has found that starvation&#x0D; and rescue sperm are adequate for fertilizing and developing embryos in sheep.

The Investigation of the Effects of Different Seminal Plasma Heating Processes on Short Term Storage of the Akkaraman Kangal Ram Sperm

Bu araştırma, koç spermasında bulunan seminal plazmanın, spermler üzerindeki etkisini incelemek ve reprodüktif açıdan kullanımını değerlendirmek amacıyla yapılmıştır. Araştırma için, 24-36 aylık yaşlarda, 8 adet ergin koçtan alınan spermalar kullanılmıştır. Spermalar çiftleşme mevsimi dışında olan koçlardan elektro ejekülatör yardımı ile alınmıştır. Spermalar seminal plazmalarından santrifüj edilerek ayrılıp, 60 ⁰C'lik ve 80 ⁰C'lik 10 dakikalık ısıtma işleminden geçirilmiştir. 60 ⁰C'lik ve 80 ⁰C'lik gruplar ile hiçbir işlem uygulanmayan kontrol gurubu 4 ⁰C'lik ortamda bekletilmeye alınmıştır. Ortalama 8 saatte bir kontrol edilen 3 grupta gözlenen motilite, canlı/ölü oranı ve spermatazoon anormallikleri kaydedilmiştir. Araştırmadan elde edilen sonuçlara göre, kontrol grubu ve deneme grupları karşılaştırıldığında en uzun süre canlılık ve motilitenin 80 ⁰C'lik grup olduğu belirlenmiştir. Bu sonuca göre spermanın seminal plazmasının içerisinde bulunan çeşitli proteinlerin ve enzimlerin denatürasyonu ile yaşam süresinin, normal spermatazoon oranının ve motilitenin doğru orantılı bir şekilde arttığını kanıtlar niteliktedir.