BackgroundProximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.MethodsThree ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).ResultsSemen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).ConclusionHigher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

BackgroundIn humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs.MethodsForty Nellore bulls were divided into three age groups: 1.8 to 2 years – young bulls; 3.5 to 7 years – adult bulls; and 8 to 14.3 years – aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA).ResultsWe report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05).ConclusionThese results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells.

The aims of this study were to evaluate cryopreserved semen of Nellore bulls of different ages and verify whether sperm quality declines with advancing age and whether lipid peroxidation and DNA damage are involved in this process. For this purpose, 40 Nellore bulls were divided into three age groups: Young, aged 1.8-2 years (n = 9); Adult, aged 3.5-7.0 years (n = 19); and Seniors, aged 8.0-14.3 years (n = 12). Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including membrane integrity, mitochondrial potential (FITC-PSA and JC1), lipid peroxidation (C-11BODIPY 581 / 591) and oxidative DNA damage (8OHdG) using flow cytometry. The thawed semen of senior bulls was characterized by a low percentage of motile sperm (33.7 ± 6.1%), higher damage to the plasma and acrosomal membrane (37.5 ± 9.8%), and low mitochondrial potential (29.1 ± 13.8%), as well as higher percentages of peroxidated cells (53.6 ± 12.2%) and DNA damage (44.1 ± 11.0%; P < 0.05). Lipid peroxidation was negatively correlated with motility (r = -0.35, P < 0.0002), average mitochondrial potential (r = -0.42; P < 0.0001) and showed a positive correlation with membrane injury and oxidative DNA damage (r = 039; P = 0.0003). Young bulls presented superior thawed sperm quality, possibly due to greater resistance to oxidative stress and, consequently, to cryopreservation. In conclusion, the sperm quality of bull semen declines with advancing age and is strongly associated with increased oxidative damage to both the plasma membrane and DNA.

Contents This report addresses an atypical transmissible venereal tumour in an 8‐year‐old bitch that was pluriparous and seropositive for leishmaniasis. There were ascites and a serosanguineous discharge from the vulva, but no lesions on the external genital mucosa. An aspirate of the peritoneal fluid showed mononuclear round cells characteristic of transmissible venereal tumour (TVT). Exploratory laparotomy revealed light red, granulomatous structures in the peritoneum, omentum, spleen, liver and uterine horns. Cytological and histopathological tests confirmed the diagnosis of intra‐abdominal TVT. Dissemination of the TVT to several organs inside the abdominal cavity probably resulted from immunosuppression caused by leishmaniasis, which favoured the presence and aggressiveness of TVT.

RESUMO: O objetivo deste estudo foi avaliar a taxa de concepção (TC) de vacas multíparas da raça Nelore apresentando diferentes escores de condição corporal (ECC), que foram submetidas ao mesmo protocolo de inseminação artificial em tempo fixo (IATF) com o emprego da gonadotrofina coriônica equina (eCG). Foram inseminadas 1574 vacas da raça Nelore, com 40 a 50 dias pós-parto. Durante as IATFs, os dados referentes ao touro (n = 8), inseminador (n = 3) e ECC foram anotados, sendo a nota do ECC aferida de 1 a 5 por técnico experiente. O diagnóstico de gestação foi realizado, por ultrassonografia, 40 dias após a IATF. Não foi observado efeito (P > 0,05) de inseminador ou touro na TC. Também não se observou diferença estatística desta taxa entre os grupos de animais separados de acordo com os ECCs. O grupo de animais com menor ECC (Grupo 1 = ECC 1,5 a 2,0; n = 139) apresentou TC de 47,4%. Os grupos de animais com ECC entre 2,5 a 2,75 (Grupo 2; n = 741) e com ECC entre 3,0 a 3,25 (Grupo 3; n = 463) apresentaram, respectivamente, TC de 47,6% e 51,2%. Os grupos de animais com maior ECC (Grupo 4 = ECC 3,5 a 4,0; n = 231) apresentou TC de 45,3% (P > 0,05). Concluiu-se que as taxas de concepção foram semelhantes entre os animais apresentando diferentes ECC no rebanho, provavelmente pelo eCG compensar a baixa pulsatilidade de LH dos animais mais magros. Porém, recomenda-se maiores estudos para verificar a real necessidade do uso de eCG em animais de condição corporal superior a 3,5.Palavras-chave: Bos indicus, ECC, fertilidade, gado de corte. DOES ECG INFLUENCE THE CONCEPTION RATE IN NELORE COWS PRESENTING DIFFERENT BODY CONDITION SCORES SUBMITTED TO THE SAME TIMED-AI PROTOCOL?ABSTRACT: The aim of this study was to evaluate the conception rate (CR) of multiparous Nelore cows presenting different body condition scores (BCS), which were submitted to the same Timed-AI protocol with equine chorionic gonadotrophin (eCG). A total of 1574 cows were inseminated, between 40 and 50 days postpartum. During insemination (timed-AI), all data regarding to bull (n=8), inseminator (n=3) and BCS (1 to 5) were recorded. The pregnancy diagnosis was performed, by ultrasonography, 40 days after timed-AI. No effect (P>0.05) of inseminator or bull was observed.No statistical difference was also observed between the groups of animals with different BCS. The