Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility / Adição de cisteína na preservação de sêmen suíno refrigerado e sua relação com a fertilidade suína

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10 percent at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65 percent of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p<0.05), when compared to the other CYS concentrations. The BTS (10.20±0.39) treated group showed a lower rate of estrus return when compared to other (BTSCYS; 86.05±039), and it showed also the highest total number of piglets borne per treatment (12.71±3.38 vs. 9.00±3.38, respectively). In conclusion, the addition of CYS in the BTS semen extender did not maintain spermatic viability of boar cooled spermatozoa and it results in a higher percentage of return to estrus and lower number of piglets borne.

A inseminação artificial é usada rotineiramente na indústria suinícula para reduzir os custos de produção além de obter maior eficiência reprodutiva durante o processo de resfriamento do sêmen. O objetivo deste trabalho foi avaliar o efeito da adição de diferentes concentrações de cisteína (CIS) ao diluidor de sêmen Beltsville Thawing Solution (BTS) resfriado sobre a qualidade espermática por até 72 horas. Foram coletados ejaculados de três cachaços e as amostras de sêmen foram diluídas em BTS, conforme os seguintes tratamentos: BTS (grupo controle); CIS0,1 (BTS + 0,1mM de cisteína); CIS0,5 (BTS + 0,5mM de cisteína); CIS1,0 (BTS + 1,0mM de cisteína); CIS2,5 (BTS + 2,5mM de cisteína); CIS5,0 (BTS + 5,0mM de cisteína); CIS10,0 (BTS + 10,0mM de cisteína) e CIS20,0 (BTS + 20,0mM de cisteína). A avaliação da integridade espermática foi determinada através de sondas fluorescentes em uma combinação de 100µg/mL FICT-PSA (isotiocinato de lecitina), 0.5mg/ml PI (iodeto de propidio), e 153µM JC-1 (5,5',6,6'-tetracloro-1,1',3,3'-tetraetillbenzimidazolil iodeto de carbocianina). As avaliações dos tratamentos foram realizadas 0, 24, 48 e 72 horas após a diluição do sêmen. Adicionalmente, foi avaliado o efeito da adição de 5,0 mM de cisteína ao diluidor BTS na manutenção da qualidade espermática e no efeito na fertilidade em suínos. Após a inseminação artificial, as fêmeas foram avaliadas quanto a taxa de retorno e o tamanho da leitegada. Durante todos os períodos analizados, os grupos CIS10,0 e CIS20,0 apresentaram menor número de espermatozóides viáveis em relação aos demais grupos. A viabilidade espermática diminuiu a níveis abaixo de 10 por cento nos tratamentos CIS10,0 e CIS20,0 nas primeiras 24 horas de armazenamento a 17ºC. Ao final do período de armazenamento todos os grupos apresentavam média inferior a 65 por cento de espermatozóides com a membrana plasmática intacta. A viabilidade espermática diminuiu significativamente quando altas concentrações de CIS (hora "0"; CIS10,0 e CIS20,0; p<0.05) foram adicionadas ao sêmen comparadas com as demais concentrações. O grupo BTS (10,20±0,39) apresentou menor taxa de retorno ao estro comparado com BTSCIS (86,05±0,39), além de apresentar maior número de leitões nascidos (12,71±3,38 vs . 9,00±3,38, respectivamente). Portanto, podemos concluir que a adição de CIS ao diluidor BTS não mantém a qualidade espermática e resulta em maior taxa de retorno ao estro e menor número de leitões nascidos.

Regulation of inducible nitric oxide synthase expression in bovine ovarian granulosa cells.

Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P<0.01). FSH (P<0.05) and IGF1 (P<0.01) stimulated oestradiol secretion and iNOS mRNA abundance in granulosa cells in vitro, whereas FGF2 (P<0.05) and EGF (P<0.01) decreased oestradiol secretion and iNOS expression. The addition of an anti-oestrogen prevented FSH-induced iNOS mRNA accumulation. Inhibition of endogenous NO production did not affect steroidogenesis in granulosa cells, but increased FasL mRNA abundance, caspase-3 activation and the incidence of apoptotic cell death (P<0.05). These results demonstrate that iNOS is expressed in ruminant granulosa cells and is regulated by gonadotrophins and oestradiol. Physiological levels of NO may contribute to the survival of granulosa cells.

Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by prostaglandins E2 and F2alpha.

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 muM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P<0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 microM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P<0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE(2), PGF(2alpha) or AngII was present in the co-culture system with follicular cells (PGE(2) 77.4%, PGF(2alpha) 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE(2) or PGF(2alpha) produced by follicular cells.