Butyrate supplementation in the liquid diet of dairy calves leads to a rapid recovery from diarrhea and reduces its occurrence and relapses in the preweaning period.

The present study aimed to evaluate the effect of continuous butyrate administration in dairy calves' liquid diet considering diarrhea, metabolic profile, gastrointestinal development, and corporal growth. Immediately after birth, calves were randomly allocated into 2 groups of 62 calves (50 females and 12 males), with access to water and a solid feed ad libitum. The butyrate group (BG) received 4 g/d of sodium butyrate (Admix Easy, Adisseo) diluted in the whole milk, and the control group (CG) received whole milk with no supplementation. Sodium butyrate was administered from d 1 of life until the weaning at 90 d. Feces consistency was assessed daily for the first 30 d of life and characterized by scores from 0 to 4 (0 and 1 for normal, and 2, 3, and 4 for abnormal feces). Diarrhea was diagnosed when the animals had abnormal feces and fever. Morbidity, recurrence, mortality, and lethality data were recorded and compared between the groups. Average daily gain (ADG) and corporal growth (body weight, thoracic perimeter, height at the withers, and croup width) were evaluated weekly, from the first day to d 30, and later at 45, 60, and 90 d of life. Blood samples were taken weekly for up to 30 d to determine the circulating levels of total calcium, phosphorus, chloride, bicarbonate, glucose, ß-hydroxybutyrate, and nonesterified fatty acids. The males were euthanized at 15 (n = 6 per group) and 30 d (n = 6 per group) for morphometric, histological, and gene expression analysis of the gastrointestinal tract. The results showed that the BG had a lower rate of morbidity (BG = 30% vs. CG = 50%) and recurrence (BG = 26.7% vs. CG = 60%) of diarrhea than the CG. In addition, the BG had abnormal feces for a shorter period (BG = 4.64 ± 0.47 d vs. CG = 8.6 ± 0.65 d). The ADG tended to be higher in BG than CG up to 30 and 60 d. Metabolic evaluations showed the lowest levels of glucose and highest levels of nonesterified fatty acids in BG. On d 30 of life, rumen papillae length, papilla area, duodenum villus length, and crypt depth were higher in BG than in CG. The duodenal gene expression at 30 d showed that animals with diarrhea episodes that did not receive butyrate had the highest levels of transcripts for the LCT and GLP2 genes. In addition, in different ways, both butyrate and neonatal diarrhea affected the gene expression of IGF1, SLC5A1, and AQP3. These results allow us to conclude that continuous supplementation with sodium butyrate improves gastrointestinal development, reduces the occurrence of diarrhea, and makes clinical conditions milder with faster recovery, favoring a higher ADG in the first 30 and 60 d of life. Based on these results, we conclude that sodium butyrate can be indicated for liquid diet supplementation to accelerate gastrointestinal tract development and prevent severe cases of neonatal diarrhea, tending to improve average daily gain until weaning.

Pharmacological approaches to induce follicular growth and ovulation for fixed-time artificial insemination treatment regimens in ewes.

In Experiment I, during the non-breeding season, after intravaginal devices containing progesterone (P4) were withdrawn (n = 28), estrous rates were greater with treatment with 400 IU eCG (P < 0.05) than with FSH (10 and 15 mg) and no treatment. During the breeding season (n = 147), estrous and pregnancy rates after fixed-time artificial inseminations (FTAI) were similar among groups: 300 IU eCG; 10 mg FSH; and control (P > 0.05). In Experiment II (non-breeding season), ewes of one group were treated with 300 IU eCG (n = 8) and of two groups were treated with 10 mg FSH. In one FSH group, 250 µg estradiol benzoate (EB) were administered after 24 h (n = 9); in the other, 4 µg GnRH were administered after 36 h (n = 10). Serum P4 concentrations were greater in eCG-treated ewes (P < 0.05). Estrous rates were similar for the eCG- and FSH plus EB-treated ewes (P > 0.05). In Experiment III (breeding season), the treatments were: 300 IU eCG; 250 µg estradiol cypionate; 250 µg EB; or control (n = 22). Follicular growth was greater for eCG-treated ewes within 0-24 h and for control ewes within 48-72 h (P = 0.001). Although estrous and ovulation rates did not differ (P > 0.05), all eCG-treated ewes had ovulations. During the non-breeding season, FSH treatment promoted follicular growth but did not induce ovulations. For FTAI regimens, eCG was more effective than FSH plus GnRH and estradiol esters in inducing estrus and ovulation.

Impact of Distinct Freezing Curves on the Quality of Ram Sperm after Thawing.

BACKGROUND: Automated equipment with customized freezing curves can be used to cryopreserve ram sperm, but none is considered a standard. OBJECTIVE: This study compared the post-thawing quality of ram sperm frozen using a conventional freezing curve and two controlled-rate curves. MATERIALS AND METHODS: Six ejaculates were collected from four rams (n = 24). In the conventional curve (110 min), sperm was cooled at -0.3 to -0.5°C min-1 until 5°C, stabilized for 60 min and exposed to liquid nitrogen (LN2) vapor for 10 min (-80°C) before submersion. The slow-customized (SC) curve (126.2 min) used a rate of -0.25°C min-1 until 5°C, stabilization for 60 min and a rate of -20°C min-1 until -120°C before immersion in LN2. Rates for the fast-customized (FC) curve (75 min) were: -0.3°C min-1 until 5°C; -3°C min-1 until -10°C; -5°C min-1 until -35°C; and -4°C min-1 until immersion in LN2 (-43°C). RESULTS: Velocity in a straight line and beat-cross frequency were lower for spermatozoa frozen with the FC than with the conventional curve (P < 0.05). The FC curve resulted in more membrane and acrosome damages than the other curves (P < 0.05). Mitochondrial membrane potential was lower with the SC than with the other curves (P < 0.05). CONCLUSION: The conventional curve was more efficient than both tested automated freezing curves. The FC curve may be an alternative to the SC curve due to the shorter processing time.

Effects of polyunsaturated fatty acids on the development of pig oocytes in vitro following parthenogenetic activation and on the lipid content of oocytes and embryos.

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.

Antioxidant Effect of Xanthan Gum on Ram Sperm after Freezing and Thawing.

BACKGROUND: Xanthan gum is used as thickener in media to preserve food products, having cryoprotectant and antioxidant properties that may be relevant for sperm cryopreservation. OBJECTIVE: To evaluate the effects of adding xanthan gum to freezing extenders on post-thawing quality and oxidant activity of ram sperm. METHODS: Ejaculates from seven rams extended TRIS-egg yolk-glycerol were split in three treatments including xanthan gum (0.15%; 0.20%; and 0.25%) and a control with no xanthan gum. RESULTS: After thawing, motility and production of reactive oxygen species (ROS) with 0.20% and 0.25% xanthan gum were lower than for the control (P < 0.05), but mitochondrial functionality and integrity of membrane, acrosome and DNA did not differ (P > 0.05). Xanthan gum at 0.20% and 0.25% may be an efficient antioxidant for frozen-thawed ram sperm, due to the reduction in ROS production.

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

Influence of cutting and deboning operations on the microbiological quality and shelf life of buffalo meat.

Considering the specific biochemical composition of buffalo (Bubalus bubalis) meat (high iron content, high biological value proteins and essential fatty acids, low amounts of fat and cholesterol), we evaluated the influence of cutting and deboning operations on the microbiological quality and shelf-life of vacuum-packed buffalo meat stored under refrigeration. On the processing day, samples were collected from carcass, deboning room surfaces and meat cuts. Samples from meat cuts were evaluated weekly for two months. On the processing day, higher counts of Pseudomonas spp. were observed in samples from meat cuts compared with the hindquarters and the processing surfaces. For thermotolerant coliform scores, the averages were -0.5 log MPN·cm(-2), -0.4 log MPN·cm(-2) and 0.9 log MPN·g(-1), respectively. Higher counts of Pseudomonas spp. and LAB in meat cuts were observed on the processing day and after the first week of storage, respectively, remaining constant during shelf life. Listeria grayi was identified in two samples of hindquarters and meat cuts during storage. Listeria innocua was identified in one meat cut. In conclusion, cutting and deboning operations influence the microbiological quality and shelf life of vacuum-packed buffalo meat stored under refrigeration.

Hazard Analysis and Critical Control Points System for a Bull Semen Production Centre.

Bull semen production centres (SPC) generally present satisfactory quality control for sperm processing, but non-standardized hygiene procedures. This study describes a Hazard Analysis and Critical Control Points (HACCP) system developed for bull SPC and subsequently implemented in a commercial SPC. After the identification of hazards at each step of semen processing and the determination of their risk and severity, monitoring and corrective procedures were designed to assess the system's efficiency. The HACCP system identified six microbiological hazards, 10 physical hazards, four chemical hazards and three critical control points. After the establishment of Good Processing Practices, Standard Operating Procedures and Standard Sanitizing Operating Procedures, the system was validated through an audit, to identify eventual failures and to define measures to correct them.

Presence of leptin and its receptor in the hypothalamus, uterus and ovaries of Swine females culled with distinct ovarian statuses and parities.

Leptin acts on energy metabolism, affecting reproductive functions through activation of its receptors in the brain and in reproductive organs. This study compared the presence of leptin and its receptor (ObR-b) in hypothalamus neurons, endometrial glands and oocytes of culled swine females across ovarian statuses and parities. Immunohistochemistry was done in samples of uterus, ovaries and hypothalamus from 28 culled females, using polyclonal antibodies antileptin and ObR-b. Immunolabelling was compared for sows categorized by parity at culling (0, 1, 2-4 and <4) and ovarian status (luteal and follicular phases of the oestrous cycle and with cysts). Immunolabelling for leptin and ObR-b in neurons and oocytes was weaker in females with cysts (p < 0.05) than in those at the follicular phase. In endometrial glands, leptin immunolabelling was less intense in females with cysts (p < 0.05), but immunolabelling for ObR-b was similar across ovarian statuses (p > 0.05). In sows culled with 2-4 parities, leptin immunolabelling in neurons and endometrial glands was more intense than in nulliparous females (p < 0.05). In comparison with sows culled at greater parities, ObR-b immunolabelling for nulliparous females was less intense in endometrial glands and in oocytes (p < 0.05), but more intense in neurons (p < 0.05). Thus, in swine, the presence of leptin and ObR-b varies across parities and is more intense in the uterus, ovaries and hypothalamus of females that were cycling before culling than in those having cystic ovaries.

Leptin and mitogen-activated protein kinase (MAPK) in oocytes of sows and gilts.

Leptin is a modulator of oocyte maturation and follicular development in swine. The MAPK are serine/threonine kinases that act as signal transduction pathways in swine ovaries. This study evaluated the presence of leptin, activated MAPK ERK 1/2 and p38 in oocytes of primordial/primary, secondary and tertiary follicles of gilts and sows. Ovaries from ten gilts and ten sows were collected in an abattoir, fixed in 10% formalin and prepared with classical histology methods. For immunohistochemistry, slides were incubated with polyclonal antibodies anti-leptin, anti-phospho ERK1/2 MAPK and anti-phospho p38 MAPK. Leptin immuno-labeling and the presence of activated ERK 1/2 MAPK were more intense for oocytes of sows (P<0.05), whereas p38 MAPK was more active for oocytes of gilts (P<0.05). Although no differences in immunolabeling for leptin and p38 MAPK were observed for oocytes of gilts at distinct follicle developmental stages (P>0.05), immunolabeling was intense for oocytes of sows included in primordial/primary follicles (P<0.05). Thus, leptin and p38 MAPK may be important to start oocyte development.

Glycerol, methyl-formamide and dimethyl-formamide in canine semen cryopreservation.

The aim of the present work was to compare the efficiency of methyl-formamide (MF), dimethyl-formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk-TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo-osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 +/- 5.4%, 61.0 +/- 7.4%, 2.9 +/- 0.5 and 57.1 +/- 5.0% for GL; 59.0 +/- 8.9%, 50.0 +/- 10.0%, 2.5 +/- 0.7 and 66.9 +/- 7.7% for MF; and 44.0 +/- 21.0%, 37.0 +/- 19.8%, 2.1 +/- 0.6 and 61.1 +/- 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 +/- 12.4%, 35.8 +/- 18.4% and 34.4 +/- 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.

Buffalo (Bubalus bubalis) pre-antral follicle population and ultrastructural characterization of antral follicle oocyte.

The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus-oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus-oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata, ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.

Protein kinase C (PKC) role in bovine oocyte maturation and early embryo development.

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

Ultrastructural and morphometric characterization of buffalo (Bubalus bubalis) ovarian preantral follicles.

The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 microm and the oocyte diameter is 24.9 microm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 microm) consist of an oocyte, with a medium diameter of 26.9 microm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 microm, and the oocyte has a mean diameter of 29.4 microm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell-oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species.

Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries.

The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 prenatral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P = 0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P = 0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular development and the growth of preantral follicles in vitro.