Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts.

Effects of adding different concentrations of melatonin (10-7 , 10-9 and 10-11  M) to maturation (Experiment 1; Control, IVM + 10-7 , IVM + 10-9 , IVM + 10-11 ) and culture media (Experiment 2; Control, IVC + 10-7 , IVC + 10-9 , IVC + 10-11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10-9  M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10-9 , IVC + 10-9 , IVM/IVC + 10-9 ). In Experiment 1, maturated oocytes from Control and IVM + 10-9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10-7 (43.5%; 56.7%) and IVC + 10-9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10-9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10-9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10-9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10-9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10-9 . Reactive oxygen species production was greater in the IVM/IVC + 10-9 treatment group. In conclusion, the 10-9  M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.(AU)

Cisteína como antioxidante para a criopreservação de sêmen de garanhões considerado de baixa congelabilidade / Cysteine as an antioxidant to frozen semen of “bad freezer” stallions

Objetivou-se avaliar o efeito da adição de cisteína (CIS), como agente antioxidante, no diluenteutilizado para criopreservação de espermatozoides equinos com histórico de sêmen com baixa congelabilidade.Três amostras seminais de sete garanhões foram obtidas por meio de vagina artificial, diluídas em meiocomercial (Botusêmen) e distribuídas em dois tratamentos: controle e CIS (2,5 mM de cisteína) ecriopreservadas. Após o descongelamento, as avaliações in vitro foram: cinética espermática computadorizada eavaliação da integridade das membranas plasmática e acrossomal por meio de sondas fluorescentes. Verificou-seque no grupo CIS o percentual de motilidade total e progressiva (23,95 ± 3,33; 4,66 ± 0,84), respectivamente, foisuperior em relação às amostras do grupo controle (11,38 ± 2,35; 2,04 ± 0,36), (P < 0,01). Não houve diferençacom relação à integridade das membranas plasmática (12,5 ± 2,6 x 15,1 ± 2,6) e acrossomal (24,2 ± 3,6 x 25,4 ±3,85), (P > 0,05), quando se comparou grupo controle e cisteína, respectivamente. Levando-se em consideraçãoque a motilidade apresenta alta correlação com a fertilidade, os resultados sugerem que a adição de cisteína naconcentração de 2,5 mM ao meio diluente antes do congelamento de sêmen incrementa a qualidade dosespermatozoides criopreservados de animais considerados de baixa congelabilidade.(AU)

The aim of this experiment was to evaluate the effect of in vitro addition of cysteine (CIS) as antioxidantused for frozen semen of "bad freezer" stallions. Three semen samples from seven stallions were obtained byartificial vagina, diluted in commercial extender (Botusêmen) and splited in two treatments: control and CIS(2.5 mM of cysteine) and cryopreserved. After post-thaw, the analyzed parameters were: computerized analysisof sperm movement characteristics and acrosmal and plasma membrane integrity by fluorescent probes. Totaland progressive motility were higher (P < 0.01) in CIS group (23.95 ± 3.33; 4.66 ± 0.84) when compared withcontrol group (11.38 ± 2.35; 2.04 ± 0.36), respectively. Regarding plasma and acrosomal membrane integritythere was no difference (P > 0.05) (12.5 ± 2.6 x 15.1 ± 2.6 and 24.2 ± 3.6 x 25.4 ± 3.85, respectively) betweenthe control and cysteine group. It is known that the motility is highly correlated with fertility so these resultssuggest that the addition of 2.5 mM of cysteine in the frozen extender medium before cryopreservation increasesthe seminal quality of "bad freezer" stallions.(AU)

Cisteína como antioxidante para a criopreservação de sêmen de garanhões considerado de baixa congelabilidade / Cysteine as an antioxidant to frozen semen of “bad freezer” stallions

Objetivou-se avaliar o efeito da adição de cisteína (CIS), como agente antioxidante, no diluenteutilizado para criopreservação de espermatozoides equinos com histórico de sêmen com baixa congelabilidade.Três amostras seminais de sete garanhões foram obtidas por meio de vagina artificial, diluídas em meiocomercial (Botusêmen) e distribuídas em dois tratamentos: controle e CIS (2,5 mM de cisteína) ecriopreservadas. Após o descongelamento, as avaliações in vitro foram: cinética espermática computadorizada eavaliação da integridade das membranas plasmática e acrossomal por meio de sondas fluorescentes. Verificou-seque no grupo CIS o percentual de motilidade total e progressiva (23,95 ± 3,33; 4,66 ± 0,84), respectivamente, foisuperior em relação às amostras do grupo controle (11,38 ± 2,35; 2,04 ± 0,36), (P 0,05), quando se comparou grupo controle e cisteína, respectivamente. Levando-se em consideraçãoque a motilidade apresenta alta correlação com a fertilidade, os resultados sugerem que a adição de cisteína naconcentração de 2,5 mM ao meio diluente antes do congelamento de sêmen incrementa a qualidade dosespermatozoides criopreservados de animais considerados de baixa congelabilidade.

The aim of this experiment was to evaluate the effect of in vitro addition of cysteine (CIS) as antioxidantused for frozen semen of "bad freezer" stallions. Three semen samples from seven stallions were obtained byartificial vagina, diluted in commercial extender (Botusêmen) and splited in two treatments: control and CIS(2.5 mM of cysteine) and cryopreserved. After post-thaw, the analyzed parameters were: computerized analysisof sperm movement characteristics and acrosmal and plasma membrane integrity by fluorescent probes. Totaland progressive motility were higher (P 0.05) (12.5 ± 2.6 x 15.1 ± 2.6 and 24.2 ± 3.6 x 25.4 ± 3.85, respectively) betweenthe control and cysteine group. It is known that the motility is highly correlated with fertility so these resultssuggest that the addition of 2.5 mM of cysteine in the frozen extender medium before cryopreservation increasesthe seminal quality of "bad freezer" stallions.

Number and density of equine preantral follicles in different ovarian histological section thicknesses.

Regardless of species, advances in preantral follicle culture and cryopreservation and transplant of ovarian tissue techniques are dependent on the number and density of preantral follicles in the ovary. This study tested the effect of different histological section thicknesses on number, classification, and density of equine preantral follicles. An ovarian fragment was obtained from 5- to 10-year-old mares (n = 14) after slaughter, and each fragment was submitted to three histological section thickness treatments: 3, 5, and 7 µm. The area (cm(2)) of each ovarian fragment was measured, and the sections were evaluated by light microscopy. The percentage of morphologically normal follicles (89%) was similar (P > 0.05) among primordial, transitional, and primary follicles and also among histological section thicknesses. A greater (P < 0.05) number of preantral follicles per histological section were seen in the 7-µm (8.0 ± 2.2) than that in the 3-µm (3.4 ± 0.7) treatment. Furthermore, a linear regression analysis reported that the number of preantral follicles increased (P < 0.05) when a thicker section treatment was used. However, no association (P > 0.05) between follicular density and treatment was observed. The mean number of preantral follicles per fragment (45.3 ± 18.8) and the follicular density (3.0 ± 0.5 follicles per cm(2)) were different (P < 0.05) among mares. In conclusion, this study on equine preantral follicles reported that (1) a 7-µm histological section thickness might be recommended because it allowed identification of a greater number of preantral follicles per sample, (2) a large individual variation in follicle population and density was detected regardless of histological section thickness, and (3) mares have a low number and density of preantral follicles when compared with those reported for other species.

Glutathione for the freezing of cooled equine semen, using different protocols

The aim of this study was to evaluate the effect of in vitro addition of glutathione in four different concentrations compared to the control group, using two different freezing systems: controlled-rate (AS) and manual (MS) freezer. The parameters evaluated were motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled (16ºC) f or 24 h. After cooling, extended semen was centrifuged at 600 xg for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender with different glutathione concentration s ( 0, 2.5 mM, 5 mM, 7.5 mM , and 10 mM). Samples were then packed into 0.5 m l straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (AS) and the other for a manual system (MS). In this study, the control group showed higher motility and better membrane integrity within treatments for the AS (P < 0.05). Furthermore, when an MS group was evaluated, the 2.5 mM glutathione group demonstrated better preservation for total motility and plasmatic membrane integrity. However, concentrations higher than 2.5 mM were deleterious to the spermatozoa in both controlled and manual freezing systems.

Glutathione for the freezing of cooled equine semen, using different protocols

The aim of this study was to evaluate the effect of in vitro addition of glutathione in four different concentrations compared to the control group, using two different freezing systems: controlled-rate (AS) and manual (MS) freezer. The parameters evaluated were motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled (16ºC) f or 24 h. After cooling, extended semen was centrifuged at 600 xg for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender with different glutathione concentration s ( 0, 2.5 mM, 5 mM, 7.5 mM , and 10 mM). Samples were then packed into 0.5 m l straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (AS) and the other for a manual system (MS). In this study, the control group showed higher motility and better membrane integrity within treatments for the AS (P < 0.05). Furthermore, when an MS group was evaluated, the 2.5 mM glutathione group demonstrated better preservation for total motility and plasmatic membrane integrity. However, concentrations higher than 2.5 mM were deleterious to the spermatozoa in both controlled and manual freezing systems.(AU)

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows.

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

The effect of offering an energy and protein supplement to grazing Canchim beef cows either postpartum or both pre- and postpartum on lipid blood metabolites and folliculogenesis.

This study was designed to evaluate the effect of nutritional supplementation offered during the pre- and postpartum periods on serum cholesterol, triglycerides and total lipids of Canchim beef cows and their relationship with folliculogenesis. Thirty cows with predicted calving date between September and October, kept in pastures of Brachiaria brizantha cv. Marandú together with their calves, were randomly distributed into three experimental groups: the first received only a mineral mixture (Control Group, CG); the second group received a concentrate with 16%crude protein/kg dry matter (DM) and 3000 kcal digestible energy/kg DM offered for 45 days prepartum and 120 days postpartum (PREG); the third group received the concentrate from parturition until the 120th day postpartum (POSG). Consumption was estimated at 1% of body weight, and each cow received approximately 4.0 kg/day (fresh weight) of supplement. Blood samples were taken and an ultrasound examination of the ovaries was performed twice a week until the 60th day postpartum. The body condition score (BCS) and the weight of the cows were recorded at 15-day intervals from calving until the 60th day postpartum. Data are presented as mean+/-SEM. Mean weight and BCS at calving were, respectively, 448+/-54.9 kg and 6.2+/-0.25 (PREG); 432+/-71.1 kg and 5.5+/-0.69 (POSG); and 434+/-66.4 kg and 5.5+/-0.69 (CG). Total cholesterol (TC), triglycerides (TRIG) and total lipids (TLIP) were measured using colorimetry until the 60th day postpartum. TC averages were PREG 186+/-62.6 mg/dL, POSG 159+/-43.1mg/dL and CG 133+/-35.1mg/dL (P<0.05). For TRIG, the means were PREG 29+/-11.3mg/dL (P<0.05), POSG 24+/-8.1mg/dL and CG 26+/-12.1mg/dL (P>0.05). Serum concentrations of TLIP were PREG 588+/-145.6 mg/dL, POSG 512+/-137.6 mg/dL and CG 452+/-122.4 mg/dL (P<0.05). The first dominant follicle (DF) was identified on Day 21+/-10.3 (PREG), 36+/-28.5 (POSG) and 51+/-32.8 (CG) after calving. The difference between PREG and CG was significant (P<0.05). TC was positively correlated with the calving to first estrus interval (P<0.05). Results showed that nutritional supplementation before parturition assured good body condition at calving and suggested that it was effective at increasing cholesterol availability to maintain ovarian follicle function and to favor earlier resumption of ovarian activity.

Granular Vulvovaginitis Syndrome in Nelore pubertal and post pubertal replacement heifers under tropical conditions: role of Mycoplasma spp., Ureaplasma diversum and BHV-1.

In order to determine the role of Mycoplasma spp, Ureaplasma diversum and BHV-1 as causal agents of Granular Vulvovaginitis Syndrome in Nelore heifers raised under tropical conditions and based on the hypothesis that stressful conditions during puberty or breeding season would be a determinant factor for the infection, 340 heifers not vaccinated against BHV-1 were divided in Post-pubertal, in the beginning of the first breeding season, and Pubertal heifers. The vaginal lesion score (VLS) Grade 1 to 4 was giving according to lesion area and severity. Vaginal mucus was used to isolate Mycoplasma spp., Ureaplasma diversum and BHV-1. The predominant VLS was 2. No sample was positive for BHV-1; 48% were positive for Mycoplasma spp., Ureaplasma diversum, or both, with predominance of Ureaplasma diversum. Serum neutralization for BHV-1 showed more positive animals in pubertal group (23%); 3 of the paired sera demonstrated seroconversion. These data indicated that post-pubertal and pubertal Nelore heifers raised under extensive conditions are more susceptible to Mycoplasma spp. and Ureaplasma diversum. The hypothesis that the stress of pubertal period could lead to an acute vaginal infection by HBV-1 was not proofed.

Effects of biostimulation and nutritional supplementation on pubertal age and pregnancy rates of Nelore heifers (Bos indicus) in a tropical environment.

To determine effects of biostimulation (BIO) and dietary supplementation (BIO+S) on pubertal age and pregnancy rates, Nelore heifers (n=392) were randomly assigned to one of four treatment groups (n=98/group). All animals were in tropical environmental conditions, in the middle-west region of Brazil, grazing in pastures of Brachiaria brizantha, cv. Marandu; Panicum Maximum, cv. Tanzânia and Brachiaria humidícula. The heifers of the BIO group were kept in the presence of bulls while being maintained on pasture; the animals in the BIO+S group were kept in the presence of bulls while being managed on pasture and were fed a diet with greater energy and protein content to produce 0.49 kg of BW gain/day; the animals in control group (the NBIO) were kept away from bulls and under pasture conditions; and the animals in the NBIO+S group were kept away from bulls, were maintained on pasture, and were fed the same diet as the BIO+S group. Heifers were bred at 22-23 months of age, and pregnancy diagnosis was made 45 days after the end of the breeding season. There were differences (P<0.05) between groups regarding pubertal heifers up to 19 months (NPH), final body weight (FBW) and pregnancy rates (P<0.01), with an advantage for the animals in the BIO and BIO+S groups. Although the effect of a diet with greater protein and energy content was not clear in this experiment, the exposure of heifers to a male during the prepubertal period decreased age at the first breeding season, resulting in a significant reduction in age of first pregnancy in Nelore heifers kept under extensive management systems in a tropical environment.

Testosterone concentration in blood serum of buffalo bulls during 24 hours in winter and summer seasons

Concentraçöes de testosterona foram determinadas por radioimunoensaio em 30 amostras de soro sanguíneo obtidas cinco vezes durante 24 horas, de 6 búfalos adultos Jaffarabadi x Mediterrâneo. As amostras foram obtidas durante um dia do inverno e um dia do veräo de 1991. As concentraçöes de testosterona variaram de 0,10 a 1,36 ng/ml no inverno e de 0,10 a 2,454 ng/ml no veräo. Valores máximos foram obtidos às 6,00 horas (0,52 ng/ml) no veräo e 0,82 ng/ml no inverno, ocorrendo entäo duas quedas abruptas, a primeira às 12 horas (0,37 ng/ml) no veräo e 0,21 ng/ml no inverno e a segunda 24 horas (0,17 ng/ml) no veräo e 0,49 ng/ml no inverno. No veräo, níveis mais altos de testosterona sérica foram observados durante o dia