Use of the flavonoid taxifolin for sperm cryopreservation from the threatened Bermeya goat breed.

Taxifolin is a plant flavonoid effective as an antioxidant. This study aimed to assess the effect of adding taxifolin to the semen extender during the cooling period before freezing on the overall post-thawing sperm variables of Bermeya goats. In the first experiment, a dose-response experiment was performed with four experimental groups: Control, 10, 50, and 100 µg/ml of taxifolin using semen from 8 Bermeya males. In the second experiment, semen from 7 Bermeya bucks was collected and extended at 20 °C using a Tris-citric acid-glucose medium supplemented with different concentrations of taxifolin and glutathione (GSH): control, 5 µM taxifolin, 1 mM GSH, and both antioxidants. In both experiments, two straws per buck were thawed in a water bath (37 °C, 30 s), pooled, and incubated at 38 °C. Motility (CASA) was assessed at 0, 2, and 5 h, and sperm physiology was assessed at 0 and 5 h by flow cytometry (viability, intact acrosome membrane, mitochondria membrane potential, capacitation, intracellular reactive oxygen species -ROS-, mitochondrial superoxide, and chromatin status). In experiment 2, an artificial insemination trial (AI) was included with 29 goats for testing the taxifolin 5-µM treatment on fertility. Data were analyzed with the R statistical environment using linear mixed-effects models. In experiment 1 and compared to the control, T10 increased progressive motility (P < 0.001) but taxifolin decreased total and progressive motility at higher concentrations (P < 0.001), both post-thawing and after the incubation. Viability decreased post-thawing in the three concentrations (P < 0.001). Cytoplasmic ROS decreased at 0 and 5 h at T10 (P = 0.049), and all doses decreased mitochondrial superoxide post-thawing (P = 0.024). In experiment 2, 5 µM taxifolin or 1 mM GSH (alone or combined) increased total and progressive motility vs. the control (P < 0.01), and taxifolin increased kinematic parameters such as VCL, ALH, and DNC (P < 0.05). Viability was not affected by taxifolin in this experiment. Both antioxidants did not significantly affect other sperm physiology parameters. The incubation significantly affected all the parameters (P < 0.004), overall decreasing sperm quality. Fertility after artificial insemination with doses supplemented with 5 µM taxifolin was 76.9% (10/13), not significantly different from the control group (69.2%, 9/13). In conclusion, taxifolin showed a lack of toxicity in the low micromolar range and could benefit goat semen cryopreservation.

Optimization of semen cryopreservation in black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguins using dimethylacetamide and dimethylsulfoxide.

Black-footed penguins (Spheniscus demersus) are classified as endangered, and the populations of gentoo penguins (Pygoscelis papua) are rapidly decreasing. The optimization of semen cryopreservation in these species, for preserving their genetic diversity in genome resource banks, is essential for the success of captive breeding programs. This study compares the effectiveness of two permeating cryoprotectants, dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), on frozen-thawed sperm characteristics. Semen samples were collected during each breeding season once a week during two consecutive years. Semen samples were packaged in 0.25 ml straws and frozen by placing them in nitrogen vapors. After thawing, sperm motility characteristics were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. DNA integrity was evaluated by TUNEL assay. Gentoo sperm characteristics after freeze-thawing did not show any differences when using DMSO or DMA. In black-footed samples, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) were greater using 8% DMSO (P < 0.05) than 6% DMA. The cryoresistance ratio (CR) using 8% DMSO was greater (P < 0.05) in gentoo than black-footed samples for CR-VCL and CR-VAP, and 6% DMA returned greater CR values (P < 0.05) than in black-footed samples for all characteristics evaluated. No differences were found in DNA fragmentation. In conclusion, the present results highlight the benefits of using 8% DMSO compared to 6% DMA in penguins. Sperm from black-footed showed a higher sensitivity to freezing-thawing process than gentoo sperm.

Effect of glutathione on pre and post-freezing sperm quality of Indian red jungle fowl (Gallus gallus murghi).

During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules and insufficient natural antioxidant defence system. Therefore, present study was designed to identify the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi) sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen was divided in four aliquots, diluted with red fowl extender (1:5) at 37 °C having GSH 0 mM (control), 0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (-196 °C) in liquid nitrogen. Semen quality was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at 37 °C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling, equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scavenging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integrity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle fowl through enriching antioxidant potential and ameliorating the oxidative stress.

Influence of testosterone administration at the end of the breeding season on sperm cryoresistance in rams (Ovis aries) and bucks (Capra hircus).

This study examines the influence of administering testosterone at the end of the mating season, on the responses (morphometric and functional) of ram and buck sperm to freezing-thawing. Five rams were administered 25 mg testosterone propionate (TP) subcutaneously in 2 mL of olive oil twice per week (Monday and Thursday) from October 1 to 31; 5 bucks received exactly the same treatment but from November 1 to 30. Control groups were administered 2 mL of olive oil without TP twice per week over the same period. In the rams, no significant differences were seen in plasma testosterone between the treated and control groups during treatment (0.8 ± 0.2 ng/mL vs 1.5 ± 0.5 ng/mL; P > 0.05). Significant differences were seen in this respect, however, in the bucks (4.3 ± 0.8 ng/mL and 6.9 ± 0.9 ng/mL; P < 0.05). In the rams, TP treatment increased (P < 0.05) the straight-line velocity (VSL), linearity (LIN), straightness (STR) and wobble (WOB) values in fresh sperm samples. Similarly, in the frozen-thawed samples, TP treatment increased the VSL, average path velocity (VAP), LIN and WOB values (P < 0.05) compared with controls. In the bucks, treatment with TP had no effect on any measured variable in fresh sperm; frozen-thawed sperm, however, returned greater VSL, LIN, STR, and WOB values (P < 0.05) than did controls. In the rams, treatment with TP led to a reduction in all fresh sperm head morphometric variables (P < 0.05). Freezing-thawing further reduced (P < 0.05) all morphometric variables in both the control and treated groups. In the bucks, treatment with TP increased (P < 0.05) the length, area, and perimeter of fresh sperm cells, unlike that seen in ram sperm. Compared with fresh sperm, freezing-thawing led to reduced (P < 0.05) morphometric variables in both the control and treated bucks, except for the sperm head width, which in the controls remained unchanged. In conclusion, TP treatment at the end of the mating season affected fresh sperm quality, in both Spanish Merino rams and Murciano-Granadina bucks, in a species-specific manner, but improved the sperm kinetic variables after freezing-thawing in both species, apparently improving sperm cryoresistance. Treatment with TP affects the dimensions of the sperm head in a species-specific manner.

Chronic use of a GnRH agonist (deslorelin) or immunization against GnRH: effects on testicular function and sperm quality of bucks.

Chronic use of GnRH agonists and immunization against GnRH have been used as reversible contraceptive methods. The aim of this study was to compare the effectiveness of both treatments to inhibit reproductive function of adult bucks, in terms of strength and duration of the effects. We used 9 control untreated bucks (CON), 7 bucks treated chronically with a GnRH agonist (subcutaneous implants with 7.4 mg of deslorelin, Suprelorin, Virbac) (AGO), and another 7 bucks were immunized against GnRH (dose of 2 mL of Improvac-Zoetis with 300 µg of a synthetic incomplete analog of natural GnRH; 300 mg of diethylaminoethyl-dextran; and 2.0 mg of chlorocresol) (IMM). Testicular and sperm evaluations, testosterone concentrations, and male odor were determined from 4 wk before applying the treatments until 17 mo of their application. Scrotal circumference of CON (21.0 ± 0.1 cm) and IMM (21.2 ± 0.2 cm) was greater than that of AGO bucks (19.9 ± 0.2 cm) (P < 0.05 for each), without difference between CON and IMM bucks. Pixels' color intensity of testicular ultrasound images was not affected by treatment (general mean ± SEM: 116.0 ± 1.8). Testosterone concentration was greater in CON than AGO and IMM in months 3 and 4, greater in CON and IMM than AGO bucks in months 15 and 16, and greater in IMM than CON and AGO bucks in month 17 (P < 0.05 for all comparisons). Male odor was greater in CON (1.5 ± 0.0) than IMM bucks (1.3 ± 0.0) and greater in IMM than AGO (1.1 ± 0.0) bucks (P < 0.05 for each). Treatment negatively affected all the sperm variables: the total number of sperm in the ejaculate, sperm motility, sperm with normal morphology and sperm with integral membrane function. It was concluded that both treatments were effective in inhibiting the reproductive axis; however, neither of them produced azoospermia or decreased testosterone concentrations to undetectable levels. With both treatments, there were individual males exhibiting characteristics of fertility in all periods of the study. However, chronic use of a GnRH agonist seemed to be the most effective treatment in terms of duration and strength.

Ovulatory activity and plasma prolactin concentrations in wild and domestic ewes exposed to artificial photoperiods between the winter and summer solstices.

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n=24) and ewes (n=24) were exposed between the winter and summer solstices to artificial long days (LD: 16 h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March ± 5 and 2 October ± 4, respectively) of cyclic ovulatory activity occurred earlier (P<0.001) than in the NP animals (26 April ± 6 and 20 October ± 2, respectively), but no differences were seen (P>0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May ± 6 and 26 April ± 6, respectively) or the onset of subsequent ovulatory activity (13 October ± 8 and 20 October ± 2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February ± 5 vs 15 March ± 11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July ± 4 vs 5 July ± 8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May ± 6, P<0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P<0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds.

Influence of vehicle on kinetics of exogenous progesterone administered either by subcutaneous and intramuscular routes to sheep.

This study aimed to determine the best vehicle and administration route for progesterone administration in sheep. In a first replicate, single intramuscular doses of 25mg progesterone were administered to ewes previously ovariectomized, either in propylene glycol (group IM-PG, n=6) or olive oil (group IM-OO, n=5). In a second replicate, the same solutions of progesterone were administered subcutaneously to the same ewes (groups SC-PG, n=6, and SC-OO, n=5). In the present study, the best pharmacokinetic results of a single dose of 25mg of progesterone were obtained, both using PG and OO as vehicles, by the subcutaneous route. Thus, progesterone remained in plasma for a longer time after subcutaneous administration in PG than in OO (t(1/2beta): 60.65+/-13.07 vs. 27.51+/-3.59 h; P<0.05); the mean residence time being higher in SC-PG than in SC-OO group (88.99+/-18.36 vs. 41.04+/-5.31h; P<0.05). However, both vehicles allowed maintained plasma levels 0.5 ng/ml for at least 30 h, so any of these treatments may be efficiently used for administration of exogenous progesterone.

New estrus synchronization and artificial insemination protocol for goats based on male exposure, progesterone and cloprostenol during the non-breeding season.

This study assesses the effectiveness of a method designed to induce and synchronize ovulation in goats during the non-breeding season, allowing for systematic timed artificial insemination (AI), without the need for prior estrus detection. This method (IMA.PRO2) induces ovulation through the "male effect" and a single 25 mg dose of progesterone given at the time of buck exposure, and early lysis of the induced corpus luteum by the administration of 75 microg of cloprostenol 9 days later. The method was tested in three separate experiments. In experiment 1, estrus was detected in 87.5% of the treated goats 37.0 +/- 1.4 h after cloprostenol administration, with the preovulatory LH surge occurring 40.5 +/- 1.6 h after the cloprostenol injection. In experiment 2, data from 503 does revealed no significant differences in fertility rates between two groups inseminated 48 h (65.5+/-4.0%) or 52 h (63+/-3.0%) after receiving cloprostenol. In experiment 3, 2184 does, comprising 37 replicate groups on 12 farms, were randomly assigned to two trial subgroups. Does in the first subgroup were treated with the IMA.PRO2 method and goats from the second group were given intravaginal progestagens for 11 days, plus 350 IU of eCG and 75 microg of cloprostenol on Day 9 of this treatment. Goats from both subgroups were cervically inseminated at the same time, 50 h after cloprostenol administration in the first group and 46 h after sponge removal in the second. The pregnancy rate achieved with the new method was 64.6%, significantly higher than the yield observed for the use of progestagens plus eCG (46.8%, P<0.01). The simple method proposed as an alternative to the use of progestagen-eCG treatment provides good pregnancy rates to AI undertaken at a fixed time point, and reduces the amount of hormone needed to synchronize estrus in the animals.

Effect of carazolol on the oestrous behaviour and concentrations of luteinising hormone and steroids in lactating cows during the periovulatory period.

Twelve multiparous, cycling, lactating Holstein-Friesian cows were synchronised with prostaglandin F(2alpha) and treated with either 2.5 mg carazolol or saline. There were no differences between the peripheral blood concentrations of oestradiol or progesterone, but in the cows treated with carazolol the periovulatory surge of luteinising hormone was delayed, and oestrous behaviour was expressed later than in the control cows.

Social dominance and breeding activity in Spanish ibex (Capra pyrenaica) maintained in captivity.

The relationship between ovulatory activity and social dominance was determined in 10 Spanish ibex females by recording their plasma progesterone and plasma cortisol levels. In a second experiment, the influence of dominance status on the establishment of pregnancy after introduction to males during late anoestrous (late October) was evaluated in another nine females. Dominance hierarchies were established in both groups by noting agonistic interactions between the individual females. Six high-ranking females of the 10 ibexes without male contact showed ovulatory activity with 1-3 progesterone cycles. The first progesterone cycle appeared in December (18 December +/- 4 days). Dominance status correlated with age (R = 0.86, P < 0.01), bodyweight (R = 0.96, P < 0.001) and the number of progesterone cycles (R = 0.82, P < 0.01). Cortisol differences were associated with differences in reproductive function rather than social status per se. Introduction to males during late anoestrous brought forward the initiation of ovulatory activity (14 November +/- 5 days) and resulted in three pregnancies in females with higher dominance ranks. The results suggest that social interactions are important in the control of ovulatory activity in the Spanish ibex.

Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats.

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.

Procedure for successful interspecific embryo transfer from mouflon (Ovis gmelini musimon) to Spanish Merino sheep (Ovis aries).

Embryos from five anesthetized mouflons (Ovis gmelini musimon), superovulated with FSH-o (Ovagen) were transferred into preselected Spanish Merino sheep (Ovis aries). Myorelaxation was complete in four of five donor mouflons. The status of the uterus of potential recipients was evaluated by transrectal ultrasonography, and those ewes with fluid in the uterine horn were rejected. The corpus luteum in each ewe was assessed ultrasonographically the day before surgery. Plasma progesterone levels and the quality of the corpora lutea were the criteria for selection of recipients. Ten embryos were transferred to the five selected Spanish Merino recipients, resulting in four pregnancies and seven live-born lambs, including three sets of twins. This study shows that determination of plasma progesterone levels combined with ultrasonographic assessment of the corpus luteum provides information useful for screening of potential recipients.

The timing of the onset of puberty, extension of the breeding season, and length of postpartum anestrus in the female mouflon (Ovis gmelini musimon).

The timing of the onset of puberty, duration of seasonal ovulatory activity, and length of postpartum anestrus were studied by means of blood plasma progesterone concentrations in a flock of European female mouflons (Ovis gmelini musimon) maintained in captivity under natural photoperiod (40 degrees 25'N). Concentrations of progesterone in the peripheral blood were determined by radioimmunoassay in samples collected from the jugular vein twice a week. First ovulations in the breeding season were highly synchronized and occurred in mid-October. In contrast, the cessation of ovulatory cycles showed significant variation among females and extended from February to May, depending on age, with 2-yr-old animals exhibiting the longest anovulatory period (P < 0.01). When lambing occurred within the breeding season (February-April), 12 out of 26 animals had their first ovulation 25 +/- 1.8 days after parturition. The 14 late-lambing females had the first postpartum ovulation delayed until the next breeding season. March/April-born mouflon lambs that reached a minimum threshold body weight (23.8 +/- 0.6 kg) in their first breeding season reached puberty at 8 mo of age. In those with slower growth rates, however, the prepubertal period was extended throughout the first breeding and nonbreeding seasons, reaching puberty during the breeding season of the following year at 19 mo of age and 27 +/- 0.3 kg body weight. Further, attainment of puberty in ewe lambs born in June/July was also delayed until the breeding season of the following year, when animals had reached a threshold body weight at 17 mo of age.

Natural infection by gastrointestinal and bronchopulmonary nematodes in mouflons (Ovis musimon) and their response to netobimin treatment.

Gastrointestinal and bronchopulmonary nematode infections and the efficacy of netobimin (Hapasil) were analyzed by way of fecal examination in 10 female mouflons (Ovis musimon), in central Spain, February 1993. Before treatment all 10 mouflons had Trichostrongylus axei, Teladorsagia circumcincta and Marshallagia spp.; sic had Nematodirus spp., two had Trichuris sp., one had Capillaria sp., seven had bronchopulmonary Dictyocaulus filaria and 10 mouflons had protostrongylid lungworms (Muellerius capillaris, Protostrongylus rufescens, Cystocaulus ocreatus or Neostrongylus linearis). Netobimin (7.5 mg/kg) was 100% effective against T. axei, T. circumcincta, Marshallagia spp., and D. filaria infections whereas one animal continued eliminating Nematodirus spp. eggs. The drug also was effective against Capillaria spp. but not against Trichuris spp. or protostrongylid infections.