RESUMO
In male goats, being in permanent visual contact with females in estrus does not prevent seasonal variation in certain endocrine hormone levels and sexual activities. In this study, we tested whether continuous and full contact with females in estrus prevented seasonal endocrinological variation in bucks. In 1 experiment (Exp. 1), we verified that the sudden introduction of goats in estrus increased the plasma concentrations of androgen in bucks during the nonbreeding season under our experimental conditions. In another experiment (Exp. 2), we tested the ability of estrous goats to prevent seasonal inhibition of LH and androgen secretions in bucks kept in permanent and full contact with them. In Exp. 1, 3 groups of bucks (n = 5 in each group) were isolated from females from the months of July to January. On January 27, one group continued being isolated from females; a second group was exposed to ovariectomized, untreated goats; and a third group was exposed to ovariectomized goats with induced estrus. Plasma androgen concentrations were determined every 2 h from 8 h before to 8 h after the introduction of females. The introduction of estrus-induced goats significantly increased androgen concentrations, which were higher than in the isolated bucks, as well as in those exposed to untreated goats (P < 0.05). In Exp. 2 (n = 5 per group), one group of bucks was isolated from females from October to July, whereas two other groups remained in contact with ovariectomized goats, either untreated or regularly induced to estrus. In the three groups of bucks, plasma concentrations of LH were determined once during the months of October, February, March, and June, whereas androgen concentrations were determined weekly from October to July. The mean plasma LH and androgen concentrations were low and did not differ among the groups of bucks during the normal seasonal period of sexual inactivity (P > 0.05). We conclude that full contact and sexual interactions with estrus-induced goats failed to stop the seasonality of LH and androgen plasma concentrations of bucks, although bucks could respond to the introduction of females by acute increases in plasma LH and androgen.
Assuntos
Androgênios/sangue , Estro/fisiologia , Cabras/fisiologia , Hormônio Luteinizante/sangue , Animais , Feminino , Masculino , Estações do AnoRESUMO
The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, µm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96â¯h of chilling were higher when the UHT extender (Pâ¯<â¯0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96â¯h compared to all other treatments (Pâ¯<â¯0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24â¯h (CS24) or chilled-for-48â¯h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3⯱â¯3.2; 48.8⯱â¯3.2; and 43.3⯱â¯3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (Pâ¯>â¯0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96â¯h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48â¯h.
Assuntos
Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Ovinos , Espermatozoides/fisiologia , Animais , Fertilização , Filtração/veterinária , Glicerol , Inseminação Artificial/veterinária , Masculino , Preservação do Sêmen/métodosRESUMO
The present study reports the effect of shortening the prefreezing equilibration time with glycerol on the quality of frozen-thawed ejaculated sperm from four Mediterranean mountain ungulates: Cantabrian chamois (Rupicapra pyrenaica), Iberian ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia). Ejaculated sperm from these species were divided into two aliquots. One was diluted with either a Tris-citric acid-glucose based medium (TCG-glycerol; for chamois and ibex sperm) or a Tris-TES-glucose-based medium (TTG-glycerol; for mouflon and aoudad sperm), and maintained at 5°C for 3h prior to freezing. The other aliquot was diluted with either TCG (chamois and ibex sperm) or TTG (mouflon and aoudad sperm) and maintained at 5°C for 1h before adding glycerol (final concentration 5%). After a 15min equilibration period in the presence of glycerol, the samples were frozen. For the ibex, there was enhanced (P<0.05) sperm viability and acrosome integrity after the 3h as compared with the 15min equilibration time. For the chamois, subjective sperm motility and cell membrane functional integrity were less (P<0.05) following 15min of equilibration. In the mouflon, progressive sperm motility and acrosome integrity was less (P<0.05) when the equilibration time was reduced to 15min. For the aoudad, the majority of sperm variables measured were more desirable after the 3h equilibration time. The freezing-thawing processes reduced the sperm head size in all the species studied; however, the equilibration time further affected the frozen-thawed sperm head variables in a species-dependent fashion. While the equilibration time for chamois sperm might be shortened, this appears not to be the case for all ungulates.
Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Glicerol/farmacologia , Preservação do Sêmen/veterinária , Ovinos/fisiologia , Animais , Criopreservação/métodos , Crioprotetores/administração & dosagem , Glicerol/administração & dosagem , Masculino , Preservação do Sêmen/métodos , Ovinos/classificação , Especificidade da Espécie , Motilidade dos Espermatozoides , TemperaturaRESUMO
A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.
Assuntos
Criopreservação/veterinária , Cabras/fisiologia , Espermatozoides/fisiologia , Animais , Criopreservação/métodos , Feminino , Fertilidade , Inseminação Artificial/veterinária , Masculino , Gravidez , Fatores de TempoRESUMO
In vitro fertilization (IVF) can be used to assess the fertilization capacity of sperm. Heterologous IVF may be useful when assessing that of wild animals as it is often difficult to obtain adequate numbers of naturally corresponding oocytes. The aim of the present study was to assess the fertilization capacity of frozen-thawed ibex epididymal spermatozoa via heterologous IVF involving the oocytes of prepubertal domestic goats. The effect on fertilization and embryo development of adding oestrous sheep serum (ESS) to the fertilization medium was also examined. Cumulus-oocyte complexes (COCs) were matured in TCM-199 for 24-27 h at 38.5°C in a 5% CO2 in air atmosphere. Frozen-thawed epididymal spermatozoa were selected by density gradient centrifugation. After maturation, the oocytes were co-incubated with spermatozoa in synthetic oviductal fluid (SOF) with different concentrations of ESS: SOF-C (0%), SOF-2 (2%) and SOF-20 (20%). At 17 h post-insemination (hpi), zygotes with one female and one male pronucleus (2PN) were categorised as normal; zygotes with 3PN were recorded as polyspermic, and oocytes with 1PN as asynchronous. Cleavage and blastocyst development were assessed at 48 and 168 hpi respectively. The percentage of zygotes with 2PN was higher in the SOF-2 than in the SOF-20 treatment group (27.7% versus 2.9% P < 0.05). The percentage of blastocysts formed with the SOF-C, SOF-2 and SOF-20 treatments were 1.1%, 7.5% and 0% respectively. These results show that the presence of 2% ESS achieves better results than the use of no serum or the standard 20% concentration. Heterologous IVF may be an effective method for predicting the fertilization capacity of ibex spermatozoa, and therefore perhaps that of other wild mountain ungulates.
Assuntos
Epididimo/citologia , Fertilização in vitro/métodos , Cabras , Técnicas de Maturação in Vitro de Oócitos/métodos , Preservação do Sêmen/métodos , Espermatozoides/fisiologia , Animais , Blastocisto/fisiologia , Criopreservação/métodos , Estro/sangue , Feminino , Fertilização , Masculino , Soro , Motilidade dos EspermatozoidesRESUMO
Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.
Assuntos
Criopreservação/veterinária , Epididimo/citologia , Glicerol , Cabras/fisiologia , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Criopreservação/métodos , Masculino , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Fatores de TempoRESUMO
The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.