l-carnitine enhances the kinematics and protects the sperm membranes of chilled and frozen-thawed Peruvian Paso horse spermatozoa.

l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through ß-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1-10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

Bicarbonate and BSA increase the capacitation pattern and acrosomal exocytosis in boar sperm after 120 min of incubation.

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence in vitro sperm capacitation in this species.

REPRODUCTIVE CYCLE STAGE ASSESSMENT IN THE TWO-TOED SLOTH (CHOLOEPUS HOFFMANNI), PRELIMINARY RESULTS.

The number of two-toed sloths (Choloepus hoffmanni) has significantly decreased in the last years. Deepening the knowledge of this tropical mammal's reproductive physiology is essential to improve captive breeding within conservation programs for this species. However, several aspects of its reproductive biology remain unexplored and have not been described sufficiently. The aim of this work was to describe the estrous cycle and reproductive physiology of two adult female C. hoffmanni by vaginal cytology, appearance of the external genitalia, and behavior. Vaginal cytology assay showed that the average duration of the estrous cycle was 15.1 ± 4.53 d. Positive correlations (P < 0.05) were found between the peak presence of superficial cells (estrous phase) and four parameters: aggressive behavior, pursuing behavior, vulvar swelling, and vaginal discharge. This pilot study, conducted on just two animals, forms a basis for a study design that may be employed for a more comprehensive assessment of the two-toed sloth reproductive physiology and behavior.

Immunolocalisation of aquaporins 3, 7, 9 and 10 in the epididymis of three wild ruminant species (Iberian ibex, mouflon and chamois) and sperm cryoresistance.

CONTEXT: In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. AIMS: We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. METHODS: Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. KEY RESULTS: This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. CONCLUSIONS: The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. IMPLICATIONS: Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing.

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Administration of carbetocin-a long-acting oxytocin analogue-before sperm collection by transrectal ultrasound-guided massage of the accessory sex glands in bucks (Capra hircus) and ibexes (Capra pyrenaica).

Transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) is a technique that allows collecting semen requiring few electrical stimuli or even no pulse. A long-acting analogue of oxytocin (carbetocin, 0.1 mg) was i.v. administered before TUMASG in 10 conscious bucks (Experiment 1) and 10 anaesthetized Iberian ibexes (Experiment 2) to shorten the time of semen collection, decrease the number of electrical stimuli and/or improve the semen quality. The ejaculated volume, concentration, quality parameters and kinetics variables of the sperm were determined in fresh semen. The time length of the procedures and the number of electric pulses applied were recorded. Furthermore, stress response indicators (number of vocalizations in Experiment 1; heart and respiratory rates, rectal temperature, cortisol levels, totals proteins and neutrophil-to-lymphocyte ratio in Experiment 2) were documented. In bucks, the administration of carbetocin tended to shorten the time needed for semen collection but no-showed differences in the fresh seminal quality. In the Iberian ibexes, there were no significant differences between groups in the time length of procedures or in the number of animals that ejaculated. Carbetocin administration only reduced the respiratory rate, did it modify fresh semen characteristics in ibexes. In conclusion, the administration of carbetocin did not appear as a useful tool to improve welfare during semen collection with TUMASG or semen quality in conscious bucks and anaesthetized ibexes, having only slight advantages related to the procedure.

Single or repeated immunization against GnRH fails to completely abolish spermatogenesis in dwarf bucks (Capra hircus).

In both captive wildlife and production animals is important to develop strategies for population control. Immunization against GnRH is an easy and inexpensive immunocastration method that reduces the concentration of testosterone and decreases sperm quality. However, its effectiveness depends on the species and repetition of the treatment. This study aimed to compare the effectiveness of a single treatment (initial immunization plus a booster with Improvac) vs repeated treatment (six doses of Improvac) to inhibit testicular function and maintain the contraceptive status during long periods in bucks. Three Dwarf bucks (Capra hircus) received two doses of Improvac, the first on Week 0, and the booster 4 weeks later (single immunization, group SI) while three Dwarf bucks received one dose of Improvac every 6 months during 3 consecutive years (repeated immunization, group RI). The other three Dwarf bucks remained untreated (control bucks, group CON). Bucks from RI had a greater decrease in scrotal circumference, testosterone concentration, male odor intensity, and sperm quality than SI bucks. However, there were no differences between SI and CON bucks in any of the variables studied. Overall, repeated treatment of Improvac decreased the testicular function of Dwarf bucks, although did not produce complete infertility. However, the repetition of the treatment produced more intensive negative effects, indicating that the strength of the effects of Improvac is rapidly lost in bucks.

Expression of Aquaglyceroporins in Spermatozoa from Wild Ruminants Is Influenced by Photoperiod and Thyroxine Concentrations.

This work identified the presence of AQPs in frozen-thawed sperm of wild ruminants and assessed the influence of the interaction between photoperiod and thyroxine on AQP expression, and on testosterone secretion. Thyroxine and melatonin were administered to ibexes. In a second experiment, performed in mouflons, circulating thyroxine was reduced via treatment with propylthiouracil (PTU), and an artificial long day (LD) photoperiod established. In the ibexes, the melatonin treatment increased the blood plasma testosterone concentration, reduced the cryoresistance ratio (CR) for sperm viability and the presence of an intact acrosome, and increased the percentage of sperm with AQP7 in the acrosome and of AQP3 and AQP10 in the midpiece. In the mouflons, neither the PTU treatment, the LD, nor the combination of both affected the CR of any sperm variable. The percentage of sperm with AQP3 increased in the post-acrosome region but decreased in the tail in the LD+PTU group. The percentage of sperm with AQP10 in the principal piece and endpiece was lower in the PTU+LD group than in the control and LD groups. The influence of photoperiod/melatonin on AQP expression might be indirectly exerted through changes in the testosterone concentration, and thus ultimately affect sperm cryoresistance.

Differential proteome between ejaculate and epididymal sperm represents a key factor for sperm freezability in wild small ruminants.

Epididymal sperm shows higher cryoresistance than ejaculated sperm. Although the sperm proteome seems to affect cell cryoresistance, studies aiming at identifying proteins involved in sperm freezing-tolerance are scarce. The aims of this study were to investigate differences of sperm freezability and proteome between epididymal and ejaculated sperm in three mountain ungulates: Iberian ibex, Mouflon and Chamois. Sperm samples were cryopreserved in straws by slow freezing. Tandem mass tag-labeled peptides from sperm samples were analyzed by high performance liquid chromatography coupled to a mass spectrometer in three technical replicates. The statistical analysis was done using the moderated t-test of the R package limma. Differences of freezability between both types of sperm were associated with differences of the proteome. Overall, epididymal sperm showed higher freezability than ejaculated sperm. Between 1490 and 1883 proteins were quantified in each species and type of sperm sample. Cross species comparisons revealed a total of 76 proteins that were more abundant in epididymal than in ejaculated sperm in the three species of study whereas 3 proteins were more abundant in ejaculated than epididymal sperm in the three species of study (adjusted P < 0.05; |log2| fold-change > 0.5). Many of the proteins that were associated with higher cryoresistance are involved in stress response and redox homeostasis. In conclusion, marked changes of sperm proteome were detected between epididymal and ejaculated sperm. This work contributes to update the sperm proteome of small ruminants and to identify candidate markers of sperm freezability.

Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant.

This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 °C) and submerging 30-µL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.

Relationship between the seasonal changes in plasma testosterone and thyroxine concentrations with sperm cryoresistance in Gabon bucks.

Gabon buck is a breed with little marked seasonality in our latitude (Uruguay, 35° SL). The role of thyroid hormones on the regulation of their seasonal reproductive activity and sperm cryoresistance is unknown. Seasonal changes in testosterone concentration can affect sperm variables, but the influence of testosterone changes on sperm cryoresistance in other species determines that the recommended time for freezing sperm does not coincide with the period with greater sperm fresh quality. The objectives of the present work were to (i) describe the thyroxine seasonal pattern in bucks in a subtropical area, and its association with annual changes in sperm variables; (ii) relate the seasonal changes of testosterone and thyroxine concentrations with the sperm cryoresistance. For one year, semen of 10 adult Gabon bucks was collected by electroejaculation every two weeks. After sperm selection, the sample was frozen. Testosterone and thyroxine concentrations varied according to the month (P < 0.0001). Testosterone reached the greatest values in April (P < 0.0001) and May (P < 0.0001) and thyroxine reached minimum values (P < 0.0001) in the same months. During these months, a negative correlation ratio (CR) was found between testosterone concentration and CR-functional membrane (R = - 0.50; P < 0.0001). CR values for most sperm variables decreased during March-May, coinciding with the presence of maximum testosterone concentrations. In conclusion, high testosterone levels are associated with the worst sperm response to freezing-thawing process. Thyroxine concentrations have a strong seasonal pattern, but there was no relationship to sperm cryoresistance.

Supplementing a skimmed milk-egg yolk-based extender with L-carnitine helps maintain the motility, membrane integrity and fertilizing capacity of chilled ram sperm.

This study examines the effect of L-carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA-FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5-15°C-2h) and at 5°C for 24 hr (LC5-5°C-24h) was tested in three ewe groups via cervical fixed-time artificial insemination. In two groups, the fertilizing capacity of the LC5-5°C-24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5-15°C-2h and LC5-5°C-24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk-egg yolk-based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.

Functional Aspects of Seminal Plasma in Bird Reproduction.

This review provides an updated overview of the seminal plasma composition, and the role of metabolic and protein components on the sperm function of avian species. In addition, the implication of seminal plasma on assisted reproductive techniques of birds was discussed. The semen of birds usually has exceptionally high sperm concentration with relatively little seminal plasma, but this contributes to very fast changes in sperm metabolism and function. The biochemical characteristics and physiological roles of the various seminal plasma components in birds (carbohydrates, lipids, amino acids, hormones, and proteins) are poorly understood. Seminal plasma content of proteins has an action on most cellular functions: metabolism, immunity, oxido-reduction regulation, proteolysis, apoptosis, ion homeostasis, and antimicrobial defenses. The variable amount of many proteins is related to a different fertility capacity of poultry sperm. The role of seminal plasma on semen conservation (chilling and freezing) remains largely a matter of speculation, as both inhibitory and stimulating effects have been found. Whereas the presence of seminal plasma did not seem to affect the sperm survival after freezing-thawing, DNA fragmentation is lower in the absence of seminal plasma. The molecular basis of the influence of seminal plasma on sperm cryo-resistance was also discussed in the present review.

Treatment of male goats with equine chorionic gonadotrophin during the non-breeding season does not affect their sperm characteristics during the subsequent breeding season.

Equine chorionic gonadotrophin (eCG) is a hormone having FSH/LH effects. It can be used to enhance sperm quality in male goats (bucks) during the non-breeding season. In a previous study carried out during the non-breeding season, we treated ten bucks with eCG (leaving nine untreated animals as control). Over a 20-day period, the treated bucks received an initial dose of 800 IU of eCG, followed by four doses of 500 IU. We found eCG enhanced semen quality, however, as also happens in female goats (does), eCG also induced a high titer of anti-eCG antibodies. In does, this lowers fertility. The aim of the present study was to determine if the eCG treatment carried out on bucks during the non-breeding season had any negative effects on their reproductive status during the following breeding season. We measured serum concentration of testosterone and anti-eCG antibody, as well as key testicular and seminal characteristics. This study commenced 91 days after the final dose of eCG in the previous study. The anti-eCG titer was higher in the treatment bucks than in untreated ones (181.7 ± 61.3 ng/µL vs 31.1 ± 10.7 ng/µL; P < 0.05). However, there were no significant differences between treated and untreated bucks in testosterone concentration, scrotal circumference, testes pixel intensity, fresh and thawed semen characteristics, or sperm cryoresistance. So, although the eCG-treated bucks had greater titers of anti-eCG antibodies, their reproductive pattern was unaffected.

UNILATERAL SINGLE VAGINAL ECTOPIC URETER WITH IPSILATERAL HYPOPLASTIC AND DEGENERATED KIDNEY ASSOCIATED WITH INFERTILITY IN IBERIAN IBEX (CAPRA PYRENAICA) DOES.

This article describes the urinogenital condition of three female Iberian ibexes (Capra pyrenaica-one infertile 3-yr-old adult and two prepubertal animals aged 1 (PP1) and 2 (PP2) yr, respectively, all raised in captivity. All showed constant urinal dribbling, leading to ulcerative dermatitis in the vulvar area. Housed in a stable with other females, the adult did not become pregnant after male contact in either of two consecutive mating seasons. Vaginoscopy and laparoscopic exploration performed on the prepubertal females revealed abnormalities of the vagina and urinary bladder. Ultrasound examination revealed atrophy of the left kidney in the adult female and PP1, and of the right kidney in PP2, with degeneration of the renal pelvis. A paraovarian cyst with hydrosalpinx was also detected in the left oviduct of the adult female. Postmortem analysis of the adult and PP2, which shared a mother, confirmed an extramural single ectopic ureter with vaginal insertion associated with atrophy of the ipsilateral kidney. Though PP1 was officially unrelated to the latter animals, all three might have had a common ancestor in their lineages.

Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep.

The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen-thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols.

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to -10 °C (5 °C/min), and then from -10 °C to -130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms.

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).

Equine Chorionic Gonadotropin (eCG) improves bucks' semen quality during the nonbreeding season.

In most goat breeds, testosterone serum concentration and semen quality decrease during the nonbreeding season. However, bucks reproductive activity may be stimulated with the administration of equine chorionic gonadotropin (eCG). Therefore, the aim of this study was to determine whether the repeated administration of eCG stimulates the reproductive status of bucks during the nonbreeding season. The study was performed with 19 bucks that were assigned to a group that was treated with eCG (GeCG) and an untreated control group (GCon). The GeCG bucks received an initial dose of 800 IU of eCG (Day 0), followed by four doses of 500 IU administered every 5 days beginning on Day 5. Serum testosterone and anti-eCG antibody concentrations, testicular and seminal traits were determined until Day 60. Testosterone concentration (from Day 3 to 21: p < 0.0001), anti-eCG titre (from Day 12 to 44: p ≤ 0.01), percentage of motile spermatozoa (Day 6: p = 0.006 and 14: p = 0.001) and of spermatozoa with progressive motility (Day 6: p = 0.01 and 14: p = 0.002) and the percentage of spermatozoa with functional membrane (Day 6: p = 0.02 and 22: p = 0.008) were higher in GeCG than in GCon bucks. Also in frozen-thawed samples, the percentage of motile spermatozoa tended to be higher in GeCG than that of GCon bucks (p = 0.07). In conclusion, the administration of eCG during the nonbreeding season stimulated the secretion of testosterone and improved fresh and possibly frozen-thawed semen quality. However, it also resulted in an increase in anti-eCG antibody titre.

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone.

The Indian red jungle fowl is a sub-species of the genus Gallus native to South Asia; facing high risk of extinction in its native habitat. During cryopreservation, permeable cryoprotectants like glycerol are usually employed and we previously showed encouraging results with 20% glycerol. Because bird spermatozoa contain very little intracellular water, the possibility of replacing an internal cryoprotectant by an external one is opened. In the present study, we tested the replacement of internal cryoprotectant glycerol by the external cryoprotectant Polyvinylpyrrolidone (PVP). PVP is a non-permeable cryoprotectant and keeps the sperm in glassy state both in cooling and warming stages without making ice crystallization within the sperm cell. We evaluated the effect of various levels of polyvinylpyrrolidone (PVP) on Indian red jungle fowl semen quality and fertility outcomes. The qualifying semen ejaculates collected from eight mature cocks were pooled, divided into five aliquots, diluted (37 °C) with red fowl semen extender having PVP [0% (control) 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v)]. Diluted semen was cryopreserved and stored in liquid nitrogen. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) with 6% PVP at post-dilution, cooling, equilibration and freeze-thawing. Higher (P < 0.05) no. of fertile eggs, fertility, no. of hatched chicks, percent hatch and hatchability was recorded with 6% PVP compared to control. It is concluded that 6% PVP maintained better post-taw quality and fertility of Indian red jungle fowl spermatozoa than glycerol and can be used in routine practice avoiding the contraceptive effects of glycerol.