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In this longitudinal study, the anti-Müllerian hormone (AMH) levels in blood were determined in 32 Murrah buffalo females at 8, 10, 12, 16 and 19 months of age when females were synchronized and the antral follicular population (AFP) was estimated. Correlations of AFP to the AMH level at 19 months of age and retrospectively to younger ages were investigated. Then females were split into high and low AFP, and their AMH levels were compared for all ages and tested as predictors of AFP categories. The highest AMH level (p < .05) was detected at 8 months, reducing but not differing (p > .05) at 10, 12 and 16 months then reducing again (p < .05) at 19 months of age. The mean AFP was 17.6 ± 6.3 follicles, and it was positively correlated with AMH in all ages tested. High AFP females had approximately two times more antral follicles than low AFP (p < .05) and their AMH levels were higher (p < .01) than in low AFP ones in all ages. Only at 8 months, AMH levels can be used to precociously detect high AFP heifers (a cut-off point of 464.7 pg/mL; p < .05), while low AFP heifers could be detected by AMH measurements at 8, 10, 12 and 16 months of age (p < .05). We conclude that AMH of buffalo calves correlates with AFP of heifers later in life and depending on the age, its levels could be used to identify future females with low or high AFP.

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O procedimento de imunocastração vem sendo utilizado como técnica alternativa amplamente favorável ao bem-estar animal, por ser indolor, pouco invasiva e com eficácia semelhante à da castração cirúrgica, que causa maior estresse aos animais, principalmente quando realizada de forma inadequada. A imunocastração estimula a produção de anticorpos contra o Hormônio Liberador de Gonadotrofina (GnRH), bloqueando temporariamente a produção da testosterona pelas gônadas masculinas. Face a escassez de informações sobre este procedimento em touros bubalinos, o estudo teve como objetivo analisar os possíveis efeitos da imunocastração no parênquima testicular de búfalos. Vinte touros bubalinos, com idade entre 2 a 3 anos, oriundos da ilha do Marajó ­ PA, foram avaliados e divididos aleatoriamente em dois grupos composto por dez animais no grupo controle (GC) e dez do grupo imunocastrado (GIM). O produto utilizado foi a vacina anti-GnRH Bopriva ® (Zoetis, SP, Brasil). O GIM recebeu duas doses de 1,0ml contendo 400µg da vacina Bopriva ® com intervalo de 8 semanas entre as aplicações e o grupo controle recebeu 1,0 ml de solução fisiológica. Após 14 dias da última dose, os animais foram abatidos e coletado os 20 pares de testículos para analises de parâmetros macroscópicos como: comprimento, largura, circunferência e peso. Além disso, foi realizada a retirada de fragmentos do parênquima testicular para confecção de lâminas histológicas. Para comparação dos dados foi aplicado o teste t de Student (Nonparametric Test) considerando a significância com p<0,05 e intervalo de confiança de 95%. Foi observado diferença significativa entre os dois grupos para grupos para peso (direto p<0001; esquerdo p≤ 0,0001), circunferência (direita p< <0,0001), 1; esquerda p<0,0001), ), largura (somente lado direito p<0,0372) e comprimento (direita p≤ 0,0013; esquerda p<0,0437). Na avaliação microscópica, os animais do GC não apresenteram alterações. Nos animais do GIM houve degeneração em todas as amostras, sendo visualizado descamação, tortuosidade e espessamento da membrana basal do túbulo seminífero, assim como vacuolização e atrofia das células de Sertoli. Foi observado também uma redução do número de células de Leydig, fibrose intertubular pronunciada, núcleos picnóticos, azoospermia e células multinucleadas no interior dos túbulos. Conclui-se a vacina anti-GnRH mostrou-se eficaz em provocar lesões no parênquima testicular, comprometendo significativamente a espermatogênese a partir da possível supressão de testosterona.(AU)

The immunocastration procedure has been used as an alternative technique widely favorable to animal welfare, as it is painless, minimally invasive and with similar efficacy to surgical castration, which causes greater stress to animals, especially when performed improperly. Immunocastration stimulates the production of antibodies against Gonadotropin Releasing Hormone (GnRH), temporarily blocking the production of testosterone by the male gonads. Due to the scarcity of information about this procedure in buffalo bulls, the study aimed to analyze the possible effects of immunocastration on the testicular parenchyma of buffaloes. Twenty buffalo bulls, aged between 2 and 3 years, from the island of Marajó - PA, were evaluated and randomly divided into two groups consisting of ten animals in the control group (CG) and ten from the immunocastrated group (GIM). The product used was the anti-GnRH vaccine Bopriva ® (Zoetis, SP, Brazil). The GIM received two doses of 1.0ml containing 400µg of Bopriva ® vaccine with an interval of 8 weeks between applications and the control group received 1.0 ml of saline solution. After 14 days of the last dose, the animals were slaughtered and the 20 pairs of testes were collected for analysis of macroscopic parameters such as: length, width, circumference and weight. In addition, fragments of the testicular parenchyma were removed for the preparation of histological slides. To compare the data, Student's t test (Nonparametric Test) was applied, considering the significance with p< 0.0372) and length (right p≤ 0.0013; left p<0.0437). In the microscopic evaluation, the animals of the CG showed no alterations. In the GIM animals there was degeneration in all samples, with desquamation, tortuosity and thickening of the seminiferous tubule basement membrane, as well as vacuolization and atrophy of Sertoli cells. A reduction in the number of Leydig cells, pronounced intertubular fibrosis, pyknotic nuclei, azoospermia and multinucleated cells within the tubules were also observed. In conclusion, the anti-GnRH vaccine proved to be effective in causing lesions in the testicular parenchyma, significantly compromising spermatogenesis from the possible suppression of testosterone.(AU)

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Heat stress reduces the reproductive capacity of bulls raised in tropical climate. However, the reestablishment of scrotal thermoregulation and the dynamics of sperm defects emergence after stress are not completely known in buffaloes. Thus, the study aimed to evaluate the effect of short-term heat stress over scrotal thermoregulation and sperm attributes, relating them to spermatogenesis stages. Five buffalo bulls went through scrotal insulation during 48 h (from day 0 to day 2). Semen samples were collected every 7 days (from day -7 to day 49) and analyzed about the progressive motility, viability, and sperm morphology. Heat stress significantly destabilized scrotal thermoregulation (P < 0.001). Scrotal temperature was from 4.2 to 6.3 °C lower than the core body temperature, except on insulation days (P < 0.001), and returned to the basal condition five days after the removal of the stressing stimulus. More significant deleterious effects were observed in sperm morphology than in cell concentration, motility, and viability. The chronology of morphologic defects expression demonstrated tail defects (days 7-14), cytoplasmic droplets (days 14-28), and head defects (day 28), returning to pre-insulation condition 35 days after the thermal challenge. Thus, hyperthermia harmed more intensely spermatozoa in epididymal transit, elongated spermatids, and secondary spermatocytes. It is concluded that water buffalo bulls present a peculiar manifestation of sperm morphology after short-term stress, indicating an important difference related to the bovine species. Therefore, during the andrological evaluation of buffalo bulls, it is necessary to avoid the allometric extrapolation between these species.

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Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

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Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.

RESUMO

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P 0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P 0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P 0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P 0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.(AU)

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RESUMO

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P 0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P 0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P 0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P 0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.

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Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

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In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

RESUMO

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

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