Correlation of maternal concentrations of plasma testosterone with fetal sex in horses / Correlação das concentrações plasmáticas de testosterona materna com o sexo fetal em equinos

ABSTRACT: The objectives of this study were to evaluate the correlation of fetal sex and plasma testosterone concentrations between the 5th and 8th months of pregnancy in mares and to verify the applicability of this test to predict fetal sex. Blood samples were collected from 21 mares at 30-day intervals of between 150 and 240 days of pregnancy. Plasma testosterone was determined by radioimmunoassay and the sex of the foals confirmed at birth. The levels of maternal testosterone were higher in mares carrying female fetuses at months 5 and 8 (P < 0.05). Limit values were determined by analyzing the receiver operating characteristic (ROC) estimates: 35.5 pg/mL and 40 pg/mL for the 5th and 8th month, respectively. For the mares with plasma testosterone values equal to or above the threshold, gestation of female foals was predicted, and for those with plasma testosterone below the threshold values pregnancy of male foals was predicted. In the 5th month, the predictive values for male and female fetuses were 70% and 88.9%, respectively; the detection rates were 87.5% and 72.7%, and the total accuracy of the examination was 78.9%. In the 8th month, the predictive values for male and female fetuses were 80% and 90%, respectively; the detection rates were 88.9% and 81.8%, and the total accuracy of the examination was 85%. It was concluded that there was a correlation between fetal sex and plasma testosterone concentrations in pregnant mares. Prediction of fetal sex based on plasma concentrations of maternal testosterone can be performed in months 5 and 8 with 78.9% and 85% accuracy, respectively.

RESUMO: Os objetivos do estudo foram avaliar a correlação do sexo fetal com as concentrações plasmáticas de testosterona entre o 5° e o 8º mês de gestação na égua e verificar a aplicabilidade deste exame para a predição do sexo fetal. Amostras de sangue foram coletadas de 21 éguas, com intervalos de 30 dias, entre 150 e 240 dias de gestação. A testosterona plasmática foi determinada por radioimunoensaio e o sexo dos potros foi confirmado ao nascimento. Os valores de testosterona materna foram superiores nas éguas gestando fetos fêmeas aos cinco e oito meses (P< 0.05). Através da análise da curva ROC (receiver operating characteristic) foram determinados valores limites de 35,5 pg/mL e 40 pg/mL para o 5º e o 8° mês, respectivamente. Éguas com testosterona plasmática igual ou acima dos valores limites foram preditas como gestando fêmeas e éguas com testosterona plasmática abaixo dos valores limites foram preditas como gestando machos. Aos cinco meses, os valores preditivos para fetos machos e fêmeas foram 70% e 88,9%, respectivamente; as taxas de detecção foram 87,5% e 72,7% e a acurácia total do exame foi de 78,9%. Aos oito meses, os valores preditivos para fetos machos e fêmeas foram 80% e 90%, respectivamente; as taxas de detecção foram 88,9% e 81,8% e a acurácia total do exame foi de 85%. Conclui-se que houve correlação entre o sexo fetal e as concentrações de testosterona plasmática em éguas prenhes. A predição do sexo fetal baseada nas concentrações plasmáticas de testosterona materna pode ser realizada aos cinco e oito meses de gestação com 78,9% e 85% de acurácia, respectivamente.

Ovine epididymal spermatozoa preservation in liquid state with or without seminal plasma epididimários ovinos / Preservação em estado líquido, com ou sem plasma seminal, de espermatozóides epididimários ovinos

ABSTRACT: Preservation and use of spermatozoa that have been recovered after death can extend the use of genetically superior animals. The objective of this study was to evaluate the maximum period for which ovine spermatozoa could be successfully stored in refrigerated dilution medium post-mortem, with or without added seminal plasma. Three samples of spermatozoa collected in an artificial vagina from 10 rams, or from the tails of four epididymes from the same rams at the time of death (G0) and six (G6), twelve (G12), twenty-four (G24) and forty-eight (G48) hours after death were used. After recovery, the spermatozoa were refrigerated at 5°C in either control medium (CM) or control medium plus 20%homologous seminal plasma (SP) and evaluated for 72 hours from the start of refrigeration. The G48 samples had a lower(P <0.05) total motility (TM) and plasma membrane integrity in the hyposmotic test (HOST) than the other groups evaluated at all analyzed times. The TM decreased (P <0.05) after 24 hours of cooling in semen collected in AV, at G0 and G24 and after 48 hours of refrigeration in G6 and G12. The TM and HOST integrity and sperm morphology did not differ between samples refrigerated in CM or SP. In conclusion, it is possible to collect epididymal spermatozoa up to 24 hours after death. Sperm viability can be prolonged fora further 48 hours by refrigeration. However, total motility decreases from 24 hours after refrigeration and the supplementation of 20% seminal plasma to the extender has no effect on spermatozoa longevity.

RESUMO: A recuperação e preservação dos espermatozoides após a morte possibilita maior aproveitamento de animais geneticamente superiores. O objetivo deste trabalho foi avaliar o período máximo após a morte do carneiro para que os espermatozoides possam ser refrigerados em meio diluidor com ou sem plasma seminal. Foram utilizadas três amostras de espermatozoides colhidos em vagina artificial (AV) de 10 carneiros ou da cauda de quatro epidídimos provenientes dos mesmos carneiros no momento da morte (G0) e seis (G6), doze (G12), vinte e quatro (G24) e quarenta e oito (G48) horas após a morte. Após a recuperação os espermatozoides (em AV ou cauda do epidídimo) foram refrigerados à 5°C em dois tratamentos: meio controle (CM) ou meio controle acrescido de 20% de plasma seminal homólogo (SP) e avaliados até 72 horas após o início da refrigeração. As amostras do G48 apresentaram motilidade total (TM) e integridade de membrana plasmática no teste hiposmótico (HOST) menor (P<0,05) que os outros grupos avaliados em todos os momentos estudados. TM diminuiu (P<0,05) após 24 horas de refrigeração no sêmen colhido em AV, no G0 e G24 e a partir de 48 horas de refrigeração no G6 e G12. A TM, integridade de membrana no HOST e morfologia espermática não diferiram entre as amostras refrigeradas em CM ou SP. Contudo, é possível refrigerar espermatozoides epididimários até 24 horas post mortem, a refrigeração prolonga a viabilidade espermática até 48 horas após o início da refrigeração. A adição de 20% de plasma seminal ao meio diluidor não tem efeito sobre a longevidade espermática.

Dois diluentes comerciais na criopreservação de espermatozoides do epidídimo de touros / Two commercial extenders for cryopreservation of epididymal bull sperm

A recuperação e a criopreservação de espermatozoides do epidídimo constituem alternativas viáveis para a preservação de material genético de animais valiosos. O objetivo deste estudo foi comparar o desempenho de dois diluentes comerciais Botu-Bov(r) (BB) e Bovimix(r) (BV), sobre a viabilidade pós-descongelação de espermatozoides do epidídimo de touros Tabapuã (Bos taurus indicus) pós-castração. Os espermatozoides foram colhidos da cauda de 20 epidídimos utilizando a técnica de fluxo retrógrado, centrifugados e diluídos com BB ou BV para posterior criopreservação a -196°C. Após a descongelação, as amostras foram avaliadas utilizando a análise computadorizada (CASA) e por análises microscópicas para a determinação da integridade de membranas plasmáticas, acrossomal e morfologia espermática. A avaliação estatística dos dados foi realizada pela análise de variância (ANOVA) com o pós-teste de comparações múltiplas de Tukey-Kramer, com nível de significância (P<0,05). Os resultados do movimento espermático avaliado pelo CASA, não diferiram para o diluente BB e BV. Também não foi observada diferença significativa entre os grupos no percentual de espermatozoides morfologicamente deformados, defeitos de acrossoma e espermatozoides com membrana plasmática íntegra após o descongelamento. Conclui-se que ambos os diluentes (BB e BV) são eficientes e podem ser utilizados na tecnologia do congelamento de espermatozoides colhidos da cauda do epidídimo de touros, não apresentando diferença na viabilidade espermática para os parâmetros estudados.

Recovery and cryopreservation of epididymal sperm is a viable alternative for preservation of genetically valuable animals. The aim of this study was to verify and to compare the effect of two commercial extenders for conventional semen on post-thawing viability of bovine epididymal sperm. For this purpose, the spermatozoa was recovered from the tail of 20 epididymis of Tabapuã bulls (Bos Taurus indicus) using retrograde flow method. After sperm recovery, the cells were centrifuged and divided for dilution with the diluents Botu-Bov(r) (BB) or Bovimix(r) (BV) for cryopreservation at -196°C. After thawing, all samples were evaluated using computer assisted sperm analysis (CASA), and by microscopic analysis for determination of integrity of plasma and acrossomal membrane and morphology. Statistical evaluation was performed by analysis of variance (ANOVA) with post-test for multiple comparisons, the Tukey-Kramer test, with significance level (P<0.05). The results of the sperm movement for diluent BB and BV evaluated with CASA, showed no difference for both (P>0.05). There was also no difference between the percentage of deformed sperm, acrosome defects and the sperm with intact plasma membrane after thawing with BB or BV. We conclude that both extenders (BB and BV) are efficient and can be used for freezing sperm collected from the epididymis of bulls, showing no difference for all the parameters studied.

The effects of intramuscular or intravenous injections of gonadotropin releasing hormone at fixed-time artificial insemination on pregnancy rates of Bos indicus beef cows

The effect of an intramuscular versus intravenous administration of gonadotropin releasing hormone (GnRH) at fixed-time AI (FTAI) on the pregnancy rates of crossbred Bos indicus beef cows was evaluated. Pluriparous nursing calv cows (n=120) were synchronized as follows: d 0 cows received a 2.0 mg injection of estradiol benzoate (EB) and insertion of a controlled intravaginal progesterone releasing device containing 0.558 g of progesterone, d 8 removal of the progesterone device , a 0.15 mg injection of prostaglandin F2α (PGF), a 1.0 mg injection of EB, and 400 IU injection of equine chorionic gonadotropin. Fifty-four hr after PGF, all cows were exposed to FTAI and a 0.084 mg injection of GnRH was administered either via Vena caudalis (n=60), or via Longissimus dorsi (n=60). Cows were inseminated with the same sire and by a single AI technician. Pregnancy was determined by the transrectal ultrasonography on d 40 after AI. Cows receiving the intravenous administration of GnRH had higher (P = 0.04) pregnancy rates than the cows receiving the intramuscular injection of GnRH (65 vs 46.6%, respectively). It was concluded that the intravenous administration of GnRH at the time of AI improved the pregnancy rates of crossbred Bos indicus beef cows submitted to FTAI.

Viability of bull spermatozoa collected from the epididymis stored at 18-20°C

The aim of this study was to evaluate the viability of bull spermatozoa collected from the cauda epididymis stored at 18-20°C, which were compared with semen collected by electro-ejaculation method and preserved at 5°C. Ten pairs of testes from Tabapuã bulls were removed by orchiectomy and stored for 6 (G6), 12 (G12), 18 (G18), 24 (G24) and 30 (G30) h at room temperature (18-20°C). Seven days before orchiectomy, semen was collected by electro-ejaculation method. The sperm parameters evaluated were: sperm motility, vigor, concentration, morphology and acrosome defects. Sperm motility declined (p<0.05) when spermatozoa were stored for 30 h in the epididymis. The spermatozoa from the epididymis showed lower sperm motility than that of spermatozoa collected via electro-ejaculation. There was a little expressive decrease in sperm vigor and increased in morphological defects with storage time, but the acrosome integrity was not affected. Cold storage (5°C) maintained sperm viable for 15 to 40.8 h. Thus, it was possible to recover viable sperm with 41.25% of motility from the cauda epididymis stored at room temperature of 18-20°C for 30 h. There were differences between the ejaculated and epididymal sperm for the bulls and the conservation at 5°C allowed short-term preservation of the gametes.

Estrus synchronization and Fixed Time Artificial Insemination (FTAI) in dairy buffaloes during seasonal anestrus

The aim of this work was to study estrus synchronization and fixed time artificial insemination (FTAI) in dairy buffaloes during season anestrus. One hundred thirty-nine dairy buffaloes in seasonal anestrus were divided in two groups as G1(n=66) and G2(n=73). The protocols for both the groups were the same until day (D)14:D0 administration of 2.0 mg estradiol benzoate and implantation of progesterone device (P4) for 14 days; D14 removal of P4 plus 150 mg of cloprostenol and 400 IU of equine chorionic gonadotropin. On D16, G1 received 10 mg of buserelin and G2 100 mg deslorelin acetate. On D17, both the groups were submitted to FTAI. Ultrasonographic examinations of ovaries were performed on D0, D14, D16 and D17. Results showed that pregnancy rates in G1 and G2 were 20 and 41% (p<0.05) and the ovulation rates were 16.6 and 37%, respectively (p<0.05). The dominant follicle (DF) diameter on D16 was 7.9 mm in G1 and 8.9 mm in G2 (p>0.05). Thirty-five percent of the animals in G1 and 54.1% in G2 showed a diameter DF greater than 8.0 mm on D16 (p>0.05). Thus, it could be concluded that the protocols synchronized the estrus, leading the concentration of the parturitions in the period of low milk production. Deslorelin was more efficient than buserelin due the higher percentage of DF ovulation and higher pregnancy rates.

Comparison between deep intracornual artificial insemination (dIAI) and conventional artificial insemination (AI) using low concentration of spermatozoa in beef cattle

This study aimed to compare the pregnancy rate using the conventional artificial insemination (AI) or deep intracornual artificial insemination (DIAI), with low number of spermatozoa (4.0 million sperm) in 270 Nelore cows. The animals were divided in two groups (G: G1 (135 cows) conventional AI was performed (=semen deposition in the uterine body) and in G2 (135 cows) to DIAI, in ipsilateral horn where the dominant follicle in the ovary had previously been detected, by ultrasound examinations. For both the methods, a single artificial insemination was carried out after visual estrus observation, checked three times a day (morning, afternoon and evening). The pregnancy diagnosis after 45 days was conducted by ultrasound. Results showed a better pregnancy rate in the DIAI group (67.4% - p<0.01), when compared to conventional AI (48.8%) with low spermatozoa concentration.

Embryo transfer in no cycling Crioula and Quarter horse breeds treated with estradiol cipionate and long-acting progesterone

The aim of this work was to prepare the mares for embryo transfer. In group 1 (G1,treated, n=15), recipient mares in anoestrus or in a transition period were treated with 5.0, 3.0 and 2.0 mg of estradiol cipionate at the days 0, 1 and 2 respectively, beginning at the day of ovulation (DO). From the fourth day on, the mares this group received long-acting progesterone weekly, up to the 120 day of gestation. At D8, the embryo was collected from the donor and transferred to the recipient. At D12, the ultrasonographyc diagnosis of pregnancy was carried out. The control group (G2, not treated, n=20) was formed by cycling recipient mares, displaying ovulation at each 2 to 3 days after the donors mare ovulation. The pregnancy rate was higher (p<0.05) in the mares from G2 (85.0 percent) than from G1 (53.3 percent). Thus, it could be concluded that the treated mares although showed lesser pregnancy rate than the cycling mare, were satisfactory alternative to be used mainly when there is no available cycling recipient.

Induction of ovulation in quarter horse mares through the use of deslorelin acetate and human chorionic gonadotrophin (hCG)

The aim this study was to compare two protocols of induction for ovulation by desloreline acetate and hCG in Quarter Horse mares. The choice of the animals was based on the observations by the estrus, by rectal palpation of the ovaries and by ultrassonography of the follicular dynamics. After estrus detection and follicle control, the measurement of the follicles and the classification of uterus were carried out. The animals that had dominant follicle (diameter more than 35 mm) and swollen uterus were used. In these conditions, the mares received hCG or desloreline acetate. Once ovulation occurred, the artificial insemination was carried. Two groups were performed: G1 (20 animals) received 1.5 mg desloreline acetate and G2 (20 animals) received 1700 IU of hCG. Following 6h intervals, the control follicular was performed by ultrasonography. The follicular average diameter was 42.6 cm for the groups and set up a score of 0 to 3 of uterine edema displayed by the device as well as the time of ovulation. In conclusion, the desloreline acetate showed better performance than hCG, because the ovulation was induced in less time (nine hours than hCG) (p<0.05).The pregnancy rate was 80 and 75 percent, respectively in G1 and G2.