Prostaglandin F2α-induced luteolysis involves activation of Signal transducer and activator of transcription 3 and inhibition of AKT signaling in cattle.

Prostaglandin F2α (PGF) induces the precipitous loss of steroidogenic capabilities and cellular death in the corpus luteum of many species, yet the molecular mechanisms underlying this event are not completely understood. Signal transducer and activator of transcription 3 (STAT3) was activated in granulosa cells during follicle atresia, whereas AKT is immediately down-regulated in the corpus luteum after PGF treatment in cattle; however, their involvement in both functional and morphological luteolysis in monovular species still need to be determined. Blood samples and corpus lutea were collected from cows before (0) and 2, 12, 24, and 48 hr after PGF treatment on Day 10 of the estrous cycle (4-5 cows per time point). Serum progesterone concentrations decreased by threefold (p < 0.05) within 2 hr, confirming functional luteolysis. The mRNA abundance of the pro-apoptotic gene BAX increased 12-48 hr post-PGF treatment (p < 0.05), while morphological luteolysis was observed 24 and 48 hr after PGF treatment, based on the loss of plasma membrane integrity, reduction of cytoplasmic volume, and pyknotic nuclei. Phosphorylated STAT3 increased, peaking at 12 hr, and remained elevated until 48 hr after PGF treatment. SOCS3 transcript abundance also increased (p < 0.05) starting at 2 hr post-PGF treatment. In contrast, AKT phosphorylation decreased by 12 hr after treatment. Thus, activation of STAT3 and inactivation of AKT signaling are involved in structural regression of the corpus luteum.

Functional status of STAT3 and MAPK3/1 signaling pathways in granulosa cells during bovine follicular deviation.

Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia.

Intratesticular hypertonic sodium chloride solution treatment as a method of chemical castration in cattle.

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control-surgically castrated immediately after birth; positive control -intact males; G1-ITI from 1- to 5-day old; G2-ITI from 15- to 20-day old; and G3-ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P<0.05), in which no testicular structure and sperm cells were observed during breeding soundness evaluation. Rectal and scrotal temperatures were not affected by different procedures. In conclusion, ITI of hypertonic NaCl solution induces sterility and completely suppresses testosterone secretion when performed during the first 20 days of life.

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.