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.

Egg Yolk-Supplemented Tris-Citric Acid Extender Improves the Prefreezing and Post-Thaw Sperm Quality Indices of Guinea Pig (Cavia porcellus) Epididymal Spermatozoa.

This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.

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.

Effect of Melatonin and Caffeine Supplementation to Freezing Medium on Cryosurvival of Peruvian Paso Horse Sperm Using a Two-Step Accelerating Cooling Rate.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 µM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

Using transrectal ultrasound-guided massage of the accessory sex glands for buck semen collection yields semen with greater cryoresistance than electroejaculation alone during the breeding season.

The cryoresistance of bucks' semen collected by transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) and electroejaculation (EE) was compared during the breeding season (Study 1) or the late non-breeding season (Study 2). Semen was collected from 10 Gabon bucks with both methods in each season and then frozen according to a standardized protocol. The time required for ejaculation, the number of electrical pulses applied, the sum of the electrical pulses applied∗voltage and the total number of vocalizations emitted during the collection by each animal were recorded. In Study 1, TUMASG required a longer time (P = 0.0006) but fewer electrical pulses and a lower sum of pulses∗voltage than EE (P < 0.0001 for both comparisons). Bucks vocalized fewer times during TUMASG than during EE (P < 0001). Semen collected with TUMASG had greater sperm concentration, sperm mass motility, total number of motile sperm and of sperm with progressive motility, and tended to have greater total number of sperm with functional membrane than semen collected with EE (P = 0.02; P = 0.003; P = 0.02; P = 0.02; P = 0.06, respectively). After the freezing-thawing process, sperm collected with TUMASG had a greater quality of the motility, a total number of motile sperm and sperm with progressive motility, a greater percentage of motile sperm and sperm with functional membrane, and tended to have a greater total number of sperm with normal morphology than semen collected with EE (P = 0.04; P = 0.04; P = 0.03; P = 0.02; P = 0.04; P = 0.06, respectively). In Study 1 sperm collected with TUMASG had greater cryoresistance for almost all the variables considered than when it was collected with EE. In Study 2, the number of electrical pulses and the sum of pulses∗voltage were greater with EE than with TUMASG (P < 0001; P = 0.0002, respectively). There were no differences in any sperm variable in fresh or thawed samples collected with TUMASG or EE. However, the other seminal characteristics evaluated did not differ according to the method, and there were no differences in the cryoresistance of any variable. Overall, TUMASG affected animal welfare less than EE and was suitable for collecting semen of good quality with high cryoresistance during the breeding season in buck.

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.

Comparison of the Conventional and Ultra-Rapid Freezing Techniques in Gabon Bucks Throughout the Year.

The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year. During 1 year, semen from 10 adult Gabon bucks was collected by electroejaculation every 2 weeks. Before and after freezing, samples were selected by density gradient centrifugation, and after sperm selection, the sample was divided into two aliquots. One aliquot was CF with an extender based on Tris, citric acid, and glucose (TCG) +6% yolk +5% glycerol, and maintained at 5°C for 3 hours of equilibration before freezing. The other aliquot was frozen using an UF method with an extender based on TCG +6% yolk +100 mM sucrose, and maintained at 5°C for 30 minutes. The evaluations included the percentages of motile sperm, sperm with progressive motility, quality of sperm motility, and the percentages of sperm with functional membrane, live sperm, sperm with morphoabnormalities, and sperm with intact acrosome. The percentage of sperm with intact acrosome was higher using the conventional freezing method (p < 0.05). After thawing and at pre- and postselection stages, the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome were greater using CF than UF (p < 0.005). Conventional freezing was more effective than UF to cryopreserve sperm from Gabon bucks, at least in our experimental conditions. Most differences in favor of CF were observed in the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome during long periods of the year, or even remained throughout it.

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.

Sperm collection by electroejaculation in small ruminants: A review on welfare problems and alternative techniques.

There are different perspectives on whether there should be use of electroejaculation (EE) for semen collection because it can be stressful and painful for the males when this technique is imposed. In the present review it is examined 1) the effects of EE on animal welfare and semen quality in domestic and wild small ruminants, 2) benefits and limitations of administering anaesthetics and sedatives prior to EE, 3) advantages/disadvantages of transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) as an alternative to EE, and 4) benefits of administering hormones, such as oxytocin or PGF2α analogues (which stimulate the contractility of the male accessory sex glands), prior to EE and TUMASG. In general, the administration of anaesthetics, sedatives or hormones reduces the pain and stress caused by EE, and can improve sperm quality, but results may vary depending on the species. The use of anaesthetics is, however, not devoid of risks and pre-EE administration of sedatives, or oxytocin or PGF2α analogues, can aid sperm collection mitigate risks. The TUMASG is less stressful than EE, but its effectiveness varies greatly among species, and it can only be performed by trained personnel. Prior administration of the hormones may also result in a reduction in the period needed to induce ejaculation with use of TUMASG procedures.

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.

Sperm collection by transrectal ultrasound-guided massage of the accessory sex glands is less stressful than electroejaculation without altering sperm characteristics in conscious goat bucks.

In anesthetized non-domestic ruminants transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) is an alternative method to collect semen slightly less stressful than electroejaculation (EE). However, some sperm characteristics are better when semen is collected with EE than with TUMASG. As anesthesia reduces the response to stressors, the advantages of TUMASG may be reduced in anesthetized animals, and thus, TUMASG may be even more advantageous in conscious animals. Therefore, the aim of the present study was to compare the stress response and the characteristics of the sperm collected with TUMASG and EE in conscious goat bucks. Semen was collected in 10 bucks with both procedures. During each procedure, the time required for ejaculation, the number of electric pulses applied and the number of vocalizations were recorded. Rectal temperature, heart rate, serum cortisol concentration, biochemical and hematological parameters were measured before and after each procedure. Sperm characteristics [ejaculated volume, sperm concentration, sperm mass motility (scale 0-5), sperm vigor (scale 0-5), the percentages of motile and progressive motile sperm, of sperm vitality, of sperm with plasma membrane integrity, and with acrosome damage and morphological abnormalities] were also determined. Electroejaculation required more electric pulses than TUMASG (P < 0.0001), but TUMASG took more time than EE (P < 0.0001). The EE provoked more vocalizations (P = 0.02) and a greater increase of cortisol concentrations than TUMASG (P = 0.04). Heart rate also tended to be greater with EE than with TUMASG (P = 0.07). The sperm characteristics did not differ between TUMASG and EE. In conclusion, TUMASG was less stressful and probably less painful than EE without affecting the semen quality. Thus, although it required more time, TUMASG is an alternative procedure to decrease the welfare concerns raised by EE in conscious goat bucks.