Addition of insulin-like growth factor I to the maturation medium of bovine oocytes subjected to heat shock: effects on the production of reactive oxygen species, mitochondrial activity and oocyte competence.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts.

Comparative efficacy of exogenous eCG and progesterone on endogenous progesterone and pregnancy in Holstein cows submitted to timed artificial insemination.

The objectives were to evaluate the effects of the administration of either eCG or progesterone (P4) alone or combined on endogenous P4 concentrations and pregnancy per AI in lactating dairy cows. Cows received a P4-releasing intravaginal device (PRID) and estradiol benzoate on D-8. The PRID was removed and a PGF2α injection was given on D-3. An estradiol cypionate was given on D-2 and TAI was performed on D0. On D-2, cows were randomly allocated to treatments in a 2×2 factorial design: Control-saline solution on the D-2 and D+3 (n=104), eCG - 400IU eCG on D-2 (n=93), P4 - 600mg of P4 on D+3 (n=106), and eCG+P4 - 400IU eCG on D-2 and 600mg of P4 on D+3 (n=95). Blood samples were collected on days three, four, and thirteen and pregnancy diagnoses were performed at 32 and 46 days after AI. There was no interaction between eCG and P4 injection. Cows treated with eCG and with P4 injection had higher serum P4 on Day +4. On Day +13 serum P4 was lower in eCG-untreated primiparous cows (Interaction eCG×parity). Cows with serum P4<4.57ng/mL on Day +13 had lower probability to be pregnant on day 32. P/AI on days 32 and 46 and embryonic losses were not influenced by eCG and P4 injection. In conclusion, the addition of 400IU of eCG on D-2 and/or 600mg of P4 on D+3 to the present TAI protocol did not increase P/AI.

Colostrum composition of Santa Inês sheep and passive transfer of immunity to lambs.

This study aimed to analyze the chemical composition and the IgG concentration of the colostrum, transitional milk, and mature milk of Santa Inês ewes as well as the transfer of passive immunity to lambs. Thirty-two pregnant ewes and 38 lambs were used. Ewes were milked immediately after lambing and at 12, 24, 36 h and 10 d postpartum. Colostrum was provided to the lambs at 40±15 min (mean±SE) after birth and then at 30-min intervals for obtaining the intake closest to 10% of body weight, and transitional milk was provided ad libitum. Blood from the lambs was collected 36 h after birth for measuring the serum concentrations of IgG, total protein, albumin, and gamma-globulin. The production was lower in primiparous than in multiparous ewes with body condition score (BCS)<2.75, but did not differ between primiparous and multiparous with BCS≥2.75 (interaction parity and BCS). The IgG concentration and fat, protein, lactose, and defatted dry extract percentages were not affected by the BCS of the ewe at lambing or by the parity. The total solids percentage in the colostrum was higher in ewes with BCS<2.75 (interaction BCS and time). The production and the protein, total solid, and defatted dry extract percentages showed quadratic behavior, the fat percentage decreased linearly, and the lactose percentage increased linearly with time postpartum. The IgG concentration in the colostrum was not correlated with the ewe's weight or BCS at the time of lambing. Moreover, the parity, the BCS, the ewe's type of gestation, and the lamb's sex did not influence the serum concentrations of IgG, total protein, albumin, and gamma-globulin in lambs. Adequate passive immune transfer (PIT) was observed in lambs for which the IgG intake was higher than 30 g. Failure in PIT was observed in 39.5% of lambs when considering a serum IgG concentration lower than 15 mg/mL and in 21% when considering a serum total protein concentration lower than 45 mg/mL. The mean apparent efficiency of absorption was 38.10%, with values between 0.02% and 98.80%. The serum IgG concentration was correlated with the total protein concentration (according to the enzymatic colorimetric method), the gamma-globulin concentration, and the absorption efficiency. The extreme variation on apparent efficiency of absorption may have an effect on the success of PIT. Lambs should consume at least 30 g of IgG in the first 24 h of life to ensure adequate PIT.

Nursing regimens: effects on body condition, return to postpartum ovarian cyclicity in Santa Ines ewes, and performance of lambs.

The effects of nursing regimens on the body condition, onset of ovarian cyclicity postpartum and weaning weight of lambs were assessed in Santa Ines ewes. Thirty-two ewes were blocked according to parity, number of lambs, and body weight at lambing and within each block randomly allocated to treatments: continuous nursing (CN), controlled nursing (CN2) with two daily feedings for an hour after the 10th day postpartum, or early weaning (EW) with total separation from the lambs after the 10th day. The animals were evaluated from the 12th day postpartum until the first estrus or until 60th day. The dry matter and nutrients intake did not differ among treatments (P>0.05) but did differ over time (P<0.01). The weight, body condition score, serum concentrations of non-esterified fatty acids and prolactin, the percentages of ewes in estrus, of ewes that ovulated within 60th day and had ovulation silent, the period from lambing to estrus, ovulation and follicle with a diameter ≥5mm and the maximum follicular diameter did not differ (P>0.05) among the treatments. The percentage of ovulation until 30th day was greater (P<0.05) in the EW group. The percentage of short luteal phases was higher in the CN2 and EW groups (P=0.07) and normal luteal phases were higher in the CN group (P=0.01). Lamb weight weaning was lower in the EW group (P<0.05). It is possible to use CN to obtain lambing periods less than eight months in Santa Ines ewes, with the advantages of simpler management and higher lamb weaning weights.

Mu opioid receptor signaling in morphine sensitization.

We used a previously reported model of morphine sensitization that elicited a complex behavioral syndrome involving stereotyped and non stereotyped activity. To identify the mechanism of these long-lasting processes, we checked the density of mu opioid receptors, receptor-G-protein coupling and the cyclic AMP (cAMP) cascade. In morphine-sensitized animals mu opioid receptor autoradiography revealed a significant increase in the caudate putamen (30% versus controls), nucleus accumbens shell (16%), prefrontal and frontal cortex (26%), medial thalamus (43%), hypothalamus (200%) and central gray (89%). Concerning morphine's activation of G proteins in the brain, investigated in the guanylyl 5'-[gamma-(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding assay, a significant increase in net [(35)S]GTPgammaS binding was seen in the caudate putamen (39%) and hypothalamus (27%). In the caudate putamen this was due to an increase in the amount of activated G proteins, and in the hypothalamus to a greater affinity of G proteins for guanosine triphosphate (GTP). The main second messenger system linked to the opioid receptor is the cAMP pathway. In the striatum basal cAMP levels were significantly elevated in sensitized animals (70% versus controls) and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) significantly inhibited forskolin-stimulated cAMP production in control (30%) but not in sensitized rats. In the hypothalamus no significant changes were observed in basal cAMP levels and DAMGO inhibition. These cellular events induced by morphine pre-exposure could underlie the neuroadaptive processes involved in morphine sensitization.