A. B. Nascimento

Progesterone supplementation after ovulation: Effects on corpus luteum function and on fertility of dairy cows subjected to AI or ET

Three experiments were done to evaluate the effects of progesterone (P4) supplementation starting during metestrus on formation of the CL and on fertility of lactating dairy cows subjected to fixed-time artificial insemination (FTAI) or embryo transfer (ET). In experiment 1, 42 Holstein cows were randomly allocated to untreated (Control) or to receive an intravaginal implant containing 1.9 g of P4 from Day 3 to 20 after FTAI (controlled internal drug release [CIDR]). Blood samples were collected on Days 3, 4, 7, 11, 14, 17, 20, and 21 after FTAI to evaluate the effect of CIDR supplementation on plasma concentration of P4 using radioimmunoassay. Ultrasound scans were performed at Days 4, 7, 11, 14, and 20 to evaluate CL volume. In experiment 2, the effect on CIDR supplementation on fertility was evaluated in 668 Holstein and crossbred dairy cows that were subjected to FTAI and allocated randomly to untreated (AI-Control) or to receive a CIDR from Day 3 to 17 (AI-CIDR) after FTAI. In experiment 3, 360 Holstein cows were treated with PGF and after heat detection (Day 0), they were allocated to untreated (ET-Control) or to receive a CIDR from Day 4 ± 1 to 8 ± 1 (ET-CIDR-4) or a CIDR from 4 ± 1 to 18 ± 1 (ET-CIDR-14). In vitro-produced embryos were transferred on Day 8 ± 1. Pregnancy diagnoses were performed by ultrasound. In experiment 1, there was interaction between treatment and day in relation to plasma P4 on Days 4 and 7 due to CIDR supplementation. Independent of treatment, pregnant cows had higher plasma P4 from Day 14 to 21 than nonpregnant cows (P ≤ 0.05). Supplementation with CIDR did not alter CL development. In experiment 2, there was no effect of supplementation of P4 on pregnancy per AI on Day 32 (32.0% vs. 31.8%, for AI-Control and AI-CIDR, respectively) or pregnancy loss (15.6% vs. 17.6%, for AI-Control and AI-CIDR, respectively). In experiment 3, P4 supplementation compromised pregnancy per ET (P/ET) on Day 32 in both supplemented groups (39.7% vs. 21.3% vs. 15.2%, for ET-Control, ET-CIDR-4, and ET-CIDR-14, respectively), with no effect on pregnancy loss. Therefore, although CIDR insertion on Day 3 after FTAI did not affect CL function and increased circulating P4, it did not increase pregnancy per AI in lactating dairy cows submitted to FTAI. Moreover, P4 supplementation decreased pregnancy per ET in lactating recipient cows.

Bovine sperm cells viability during incubation with or without exogenous DNA

SummaryThe aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 106/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Development of insulin resistance in dairy cows by 150 days of lactation does not alter oocyte quality in smaller follicles

The objective of this study was to test the hypothesis that high-producing dairy cows become increasingly resistant to insulin throughout lactation and that, consequently, oocyte quality is compromised. We used Holstein cows at 50 (51.5±3.7; n=30), 100 (102.3±9.4; n=30), and 150 (154.5±18.9; n=30) days in milk (DIM). We measured circulating insulin and glucose and performed a glucose tolerance test (GTT) after 5h of fasting. To evaluate oocyte quality, we performed ovum pickup on the day before the GTT (581 oocytes). We performed statistical analyses using the MIXED procedure of SAS. The model included the fixed effects of DIM, period, time, parity, and an interaction between DIM and time. We observed no difference in the GTT between groups for any variable related to circulating glucose (for example, glucose peak=203.3±7.2, 208.8±6.3, and 194.3±5.9mg/dL). However, various measures of circulating insulin were different in cows at 150 DIM compared with 50 or 100 DIM: higher basal insulin (8.8±0.9, 8.8±0.8, and 11.9±0.8 µIU/mL), peak insulin (61.9±6.2, 69.1±5.7, and 89.0±6.1 µIU/mL), delta maximum insulin (51.1±5.5, 59.4±5.0, and 73.5±5.4 µIU/mL), and area under the curve 5-60 (1,874.8±171.0, 2,189.5±157.8, and 2,610.5±174.0 µIU/mL × min). Nevertheless, we observed no difference among groups in the number of viable oocytes (3.2±0.7, 3.9±0.7, and 3.6±0.7 per cow per ovum pickup) or percentage of viable oocytes (49.3, 52.2, and 51.8%). Increased circulating insulin before and throughout the GTT in cows at 150 DIM indicates that cows develop increasing insulin resistance with increasing DIM; however, increased insulin resistance was not associated with a detectable alteration in the quality of oocytes aspirated from small and medium-sized follicles.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX- blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection

This experiment aimed to compare circulating progesterone (P4), follicular dynamics, and fertility during reuse of intravaginal P4 implants that were sanitized by autoclave or chemical disinfection in lactating Holstein cows submitted to fixed-time artificial insemination (FTAI). For this, 123 primiparous and 226 multiparous cows from 2 farms, averaging (mean ± standard deviation) 163.9 ± 141.9 d in milk, 35.7 ± 11.3 kg of milk/d, and a body condition score of 2.9 ± 0.5, were enrolled in the study. Cows were randomly assigned to 1 of 2 treatments using a completely randomized design and each cow received a reused implant (1.9 g of P4; previously used for 8 d) that was either autoclaved (AUT; n = 177) or chemically disinfected (CHEM; n = 172) on d -10. Also on d -10, cows received 2 mg of estradiol benzoate and 100 μg of GnRH. On d -3, cows received 25 mg of dinoprost (PGF2α). A second PGF2α was given on d -2, along with 1 mg of estradiol cypionate and P4 implant removal. Cows received FTAI on d 0. A subset of cows (n = 143) was evaluated by ultrasound on d -10, -8, -6, -3, -2, 0, and 5 to identify ovarian structures, and blood was sampled on d -10, -3, and -2 for P4 concentrations by RIA. Pregnancy diagnoses were performed at d 32 and 60. Statistical analyses was performed using PROC-MIXED for continuous variables and PROC-GLIMMIX of SAS 9.4 (SAS Institute Inc., Cary, NC) for binomial variables. The treatments did not differ in circulating P4 on d -10 or -3, but P4 was greater on d -2 in CHEM cows. Ovulation to the treatments on d -10 was associated with lower circulating P4 on d -10 (2.0 vs. 3.1 ng/mL) and resulted in greater P4 on d -3 (4.0 vs. 2.4 ng/mL) and more cows with a corpus luteum on d -3 (100 vs. 40%) than nonovulating cows. Cows that ovulated to d -10 treatments were more likely to have a synchronized new follicular wave (97.9 vs. 63.2%) and had an earlier wave emergence (1.9 vs. 2.6 d), resulting in less cows ovulating a persistent follicle (0.0 vs. 35.7%). Type of P4 implant, corpus luteum presence on d -10, and ovulation to d -10 treatments did not affect fertility (pregnancy per AI; P/AI). However, P/AI on farm A was greater than on farm B at 32 (40.8 vs. 27.8%) and 60 d (35.8 vs. 24.3%), independent of treatment. In conclusion, P4 implants with different P4 release patterns did not produce detectable differences in follicular dynamics, synchronization rate, or P/AI. Nevertheless, presence of corpus luteum or ovulation at the beginning of the FTAI protocol affected reproductive variables, such as timing and synchronization of follicular wave emergence, and size of the ovulatory follicle. Beyond that, more overall synchronized cows became pregnant to the FTAI protocol.

306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 µg mL-1), LH (0.5 µg mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 µg mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrodes buffered medium, mTBM) and placed in petri dishes containing 50 µL of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17°C. It was then centrifuged at 1200g for 3 min and standardized for 1 × 105 spermatozoa mL−1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5°C and 5p CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 µL), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1p orcein in 45p acetic acid and evaluated under phase-contrast microscopy at a 400× magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 ± 9.6p (162/412), and no difference was observed in comparison with the T2 group, 29.5 ± 4.9p (113/383). The monospermic penetration rate was 31.5 ± 6p (51/162) in the T1 group and differed from that in the T2 group, 71.7 ± 3.3p (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 ± 6p (111/162), compared with the T2 group, 28.3 ± 3.3p (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP p PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.

345 Influence of different maturation media on in vitro swine embryo development.

The aim of this work was to study the influence of maturation media NCSU23, Whitten, and TCM199 on in vitro development of swine embryos. Oocytes from slaughtered gilt ovaries were in vitro-matured (IVM) with NCSU23 (n = 148), Whitten (n = 151), and TCM199 (n = 170) media for 44 h. After IVM, all oocytes were fertilized in mTBM medium for 6 h and cultured in NCSU23 with 0.4% BSA for 5 days. After Day 5, all embryos were cultured in NCSU23 supplemented with 20% FCS for 4 days. Cleavage, blastocyst, and hatching rates were evaluated. Cleavage rates were 29% for NCSU23, 35.8% for Whitten, and 42.3% for TCM199 maturation media, with no significant difference among media (P > 0.05). Blastocyst rates were similar (P > 0.05), 18.9%, 21.8%, and 20%, respectively, for NCSU23, Whitten and TCM199 maturation media. Hatching rates were 16.1%, 34.3%, and 32.3%, respectively, for NCSU23, Whitten, and TCM199 maturation media, with no significant differences (P > 0.05). In conclusion, all three maturation media supported IVF, allowing embryo development from cleavage until blastocyst hatching.

228 Collection and evaluation of semen of sloth (Bradypus tridactylus).

Sloths are animals that suffer with the destruction and fragmentation of forests. They experience a low population growth rate and need to be studied further for the preservation of the species. The objective of this study was to contribute data relevant to the reproductive physiology of this species, selecting a semen collection method and evaluating seminal characteristics that have never before been described in the literature. Fifteen Bradypus tridactylus males were captured in Manaus, Brazil. Nine of them were captured during the first half of 2004 (Group 1) and the others during the second half (Group 2). The animals were anesthetized with an i.m. injection of a combination of ketamine (10 mg/kg) and xylasine (1 mg/kg). Semen was collected by electroejaculaton using a rectal probe designed for domestic cats. Electrostimulations were given with a 0-100 mA/0-12 V variable electrostimulator in sequences of three progressive intensities, with ten repetitions at each intensity and variation of 10 mA between them. They started with 20 mA and peaked at 60 mA. Each stimulus lasted about 3 s. It was not possible to define the best intensity of stimulus to use and ejaculation could take place at any time of the stimulation (Fishers exact test). Sperm motility and vigor were immediately analyzed. Sperm count was determined in a Neubauer chamber at a 1:50 (v:v) dilution in formol-saline. Morphology was examined at the same dilution. Fresh semen smears were made and stained using Spermac Stain® (Minitub, Tiefenbach, Germany) protocol for a better evaluation of the spermatozoa acrosome and midpiece. In both methods 200 cells were counted for morphological evaluation. All animals ejaculated approximately 30 ¼L to 90 ¼L of semen. In some ejaculates the semen was too thin and flowed down the penis, so that the volume effectively collected was not sufficient for a complete spermiogram. Spermatozoa presented a wide variety of defects, and some physical characteristics differed (not significantly) between samples collected during the first and second halves of the year. Motility and vigor were very low, the sperm did not show forward progression, only oscillatory movement. However, a high percentage (80%) of spermatozoa were moving. The concentration in Group 1 ranged from 5000 spermatozoa/mm3 to 685 500 spermatozoa/mm3 (mean ± 218 571.4 ± 242 499.4). Sperm concentation was not assessed in Group 2. The morphology of the head could be elongated or squared, or the head could have a base narrower than the apex. The tail showed a unique feature: the midpiece narrowed abruptly, forming a nip in its transition to the tail. This was similar in appearance to the segmental aplasia of the mitochondrial sheath, but it was considered normal because it was observed in all spermatozoa. Although further studies are necessary to standardize the semen evaluation of sloths and to define the best protocol for electroejaculation, this pioneering study has shown the characteristics of sloth spermatozoa and the possibility of collecting semen throughout the electroejaculation process in this species. This work was supported by Fapesp 03/07457-4.