Superovulated Cows Research Articles

Association between metabolic parameters and embryo production in superovulated dairy cattle.

This study aims to investigate the relationship between metabolic parameters and the number of embryos produced in superovulated cows with high genetic characteristics in milk yield. Eighteen Holstein donors were treated with classic superovulation protocols, AI and flushing. During superovulation, decreasing doses of FSH (follicle-stimulating hormone) were administered at 12-h intervals for 4 days. Plasma insulin-like growth factor (IGF1), glucose (GLU), beta-hydroxybutyric acid (BHB), non-esterified fatty acid (NEFA), blood urea nitrogen (BUN) and total protein (TP) levels were determined by using an autoanalyzer. The mixed model analysis of variance was used for statistical analysis. As a result, plasma IGF1, BHB and BUN had significant interactions with both groups and days (p < .05). Additionally, plasma TP-days interactions were significant (p < .05). Furthermore, there was a negative correlation between the number of embryos and plasma BHB levels (p < .05). In conclusion, under appropriate environmental conditions, metabolic profile control of donors can contribute to the embryo production process and to the studies on the metabolic infrastructure.

Polymorphisms within the &lt;em&gt;IGF1&lt;/em&gt; and &lt;em&gt;IGF1R&lt;/em&gt; genes associated with superovulation-related traits in Holstein dairy cows managed in a semiarid environment

Embryo transfer (ET) is an artificial reproductive technology used for the genetic improvement of cattle. High variation has been observed in superovulation (SPO) and embryo flush recovery, which appear to be influenced by donor cow genetics. Then, the objective was to identify single nucleotide polymorphisms (SNPs) from the genes IGF1 and IGF1R associated with reproductive traits related to SPO response in dairy cows raised in a semiarid region. Sixty-four Holstein cows were subjected to SPO, artificial insemination, and nonsurgical embryo collection. Individual blood samples were collected and used to genotype 13 SNPs from the genes IGF1 and IGF1R. Additional blood samples were collected to measure anti-Mullerian hormone (AMH) concentrations. Ovarian and embryo traits related to SPO response were evaluated. A mixed-effects model was used to identify associations between SNPs and SPO-related traits. A regression model was implemented to calculate allele substitution effects. From 13 SNPs, the SNP rs109763947 in the gene IGF1 and the SNPs rs110343126 and rs208140993 in the gene IGF1R were predictors for six traits evaluated in superovulated cows. The most favorable genotypes for these SNPs were CC, AA and GG, respectively. A linear trend was detected, suggesting an additive effect of the genes. Moreover, all traits evaluated in the current study improved as the number of favorable SNP genotypes increased, confirming a positive contribution of the SNP genes. In conclusion, three SNPs in the genes IGF1 and IGF1R were marker predictors for reproductive traits related to SPO response and embryo production in Holstein cows managed in a semiarid region.

Administração de prostaglandina F2 alfa no momento da inseminação artificial não afeta a produção de embriões bovinos

ABSTRACT: Treatment with prostaglandin F2α (PGF) induces ovulation and increases conception rates in cows, while improving embryo production in buffalos. However, its effect on superovulated cows is unknown. This study verified whether single PGF administration concurrent with artificial insemination (AI) improves fertilization and embryo production rates in superovulated cows. In each replicate, embryo donor cows were equally allocated to two groups: the untreated control and PGF groups. The latter of which received 482 µg of cloprostenol concurrent with the first AI. Each cow (n = 35) was subjected to two superovulations (SOV) in a crossover design (total = 70 embryo collections). In the control and PGF groups, respectively, the observed responses were [median (95% CI)]: 12 (10-18) and 15 (12-18) total structures, 9 (7-11) and 7 (6-10) viable embryos, 1 (0-1) and 1 (1-3) degenerated embryos, and 1 (0-3) and 2 (0-5) oocytes (P &gt; 0.05). In conclusion, single PGF treatment concurrent with the first AI did not affect embryo production in superovulated cows.

152 An effective method of inducing oestrus in superovulation-treated Japanese Black donor cows after egg collection

The objective of this study was to evaluate the effect of inducing oestrus in superovulation-treated Japanese Black donor cows after egg collection using a controlled internal drug release (CIDR) device and prostaglandin F2α treatments. The test animals were 50 biparous or triparous Japanese Black cows that had been treated for superovulation during the period of 75-110 days post-calving and from each of which 10 or more ova or embryos had been recovered using a uterine reflux method. In the 25 cows of the control group that received the conventional treatment, a CIDR was not introduced into the vagina, the number of days from egg harvesting to standing oestrus was recorded, and AI was performed using frozen semen. At the time of AI, 50µg of gonadotrophin-releasing hormone (GnRH; Consultan, Aska Animal Health) was administered intramuscularly. The cows that did not conceive in the first AI attempt were again given the same AI and GnRH treatments in the next oestrus cycle. In the 25 cows of the treatment group, a CIDR (CIDR 1900, Zoetis) device was inserted in the vagina after egg harvesting and was kept there for 12 days. At the time of insertion and removal of the CIDR, 150µg of d-cloprostenol formulation (Dalmazin, Kyoritsu Seiyaku) was administered intramuscularly. The number of days from egg harvest to standing oestrus was recorded, and GnRH was administered at the time of AI. The cows that did not conceive in the first AI attempt were given the same AI and GnRH treatments in the next oestrus cycle. The control and treatment groups were compared for the number of days from egg harvesting to oestrus (t-test) and the conception rate after AI (chi-square test). The mean number of days from egg collection to standing oestrus in the control group was 38.6±11.9, and the conception rate was 60% (15/25). Among the animals that failed to conceive in the first AI attempt, the second standing oestrus occurred 60.7±13.1 days after egg collection, and 70% (7/10) of the cows conceived after the second AI. In the treatment group, on the other hand, the mean number of days from egg collection to standing oestrus was 15.8±3.9, and 72% (18/25) of the cows conceived after the first AI. Among the cows that failed to conceive, the second standing oestrus occurred 37.4±4.7 days after egg harvest, and 71% (5/7) of the cow conceived after the second AI. There was a significant difference between the two groups in the number of days from egg collection to the first standing oestrus (P&amp;lt;0.05). The results described above suggest that keeping a CIDR in the vagina for 12 days and intramuscularly administering prostaglandin F2α at the time of insertion and removal of the CIDR in superovulated cows are effective for early onset of standing oestrus after egg collection and thus can reduce the number of days to conception.

PSXIII-13 Artificial insemination by estrus detection or by fixed-time in superovulated beef cows.

PSXIII-13 Artificial insemination by estrus detection or by fixed-time in superovulated beef cows.

Effect of insulin or insulin-like growth factor-I administration at mid-luteal phase of the estrous cycle during superovulation on hormonal profile of Sahiwal cows.

Aim:The present study was designed to study the effect of insulin and insulin-like growth factor-1 (IGF-I) administration during luteal phase of the estrous cycle on the hormonal profile of Sahiwal embryo donor cows during superovulation.Materials and Methods:Cows (n=18) were selected and divided into three groups; control (n=6, untreated), T-I (n=6, insulin-treated), and T-II (n=6, IGF-I treated). Insulin and IGF-I were given S/C on 5th, 6th, 7th, and 8th days of estrous cycle. Superovulatory treatment was started on day 9th of the estrous cycle. With the sixth dose of follicle-stimulating hormone, prostaglandin was injected to induce superovulatory heat. The superovulated cows were bred, and superovulatory response of each animal was recorded. The embryos were collected non-surgically on the 7th day of superovulatory estrus. About 15 ml blood without anticoagulant was collected on days 5, 7, 9, 11, 13, 15, 17, 19, and 21 or day of embryo recovery where day 0 of estrous cycle was taken as day of estrus. Serum was separated, centrifuged, and transferred into sterilized serum vials. All samples were stored at −20°C till analysis. Progesterone, insulin, and IGF-I were estimated in blood serum by radioimmunoassay using radioimmunoassay kits.Results and Discussion:The mean concentration of progesterone on days 7 and 11, insulin on days 7 and 9, and IGF-I on days 5, 7, 9, 11, and 15 was significantly higher in insulin-treated T-I groups as compared to untreated control.Conclusion:It may be concluded that exogenous insulin administration during mid-luteal phase may be helpful in follicular and embryonic development through modulation of progesterone, insulin, and IGF-I in indigenous (Bos indicus) Sahiwal embryo donor cows.

Day-7 embryos generate an anti-inflammatory immune response in peripheral blood immune cells in superovulated cows.

Recently, we demonstrated both in vitro and in vivo that an immunological crosstalk between Day-7 embryo and immune cells exists locally in the uterus in cows. The peripheral immune response to early embryos at Day-7 of pregnancy in cows remains largely unknown. Thus, we aimed to investigate the response of peripheral blood immune cells in the presence of multiple Day-7 embryos in the uterus in donor cows of embryo transfer program. Superovulated cows were either inseminated (n=13) at 12-18hours post-estrus or remained without inseminations (n=6). Blood was collected following insemination (Day-1) and immediately after uterine flushing (Day-7). Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood for analysis of gene expression. Interferon-stimulated genes (ISGs: ISG15 and OAS1) were increased in both PMNs and PBMCs, with up-regulation of PTGES and anti-inflammatory cytokines (TGFB1 and IL10) expressions at Day-7 of post-inseminations in cows, when compared to those at Day-7 in non-inseminated cows. The findings indicate that the presence of multiple embryos in the uterus generates an anti-inflammatory immune response in peripheral blood immune cells at Day-7 of pregnancy in cows, which might help to accept semi-allogenic embryo in the uterus.

Equine Chorionic Gonadotropin Modulates the Expression of Genes Related to the Structure and Function of the Bovine Corpus Luteum.

We hypothesized that stimulatory and superovulatory treatments, using equine chorionic gonadotropin (eCG), modulate the expression of genes related to insulin, cellular modelling and angiogenesis signaling pathways in the bovine corpus luteum (CL). Therefore, we investigated: 1—the effect of these treatments on circulating insulin and somatomedin C concentrations and on gene and protein expression of INSR, IGF1 and IGFR1, as well as other insulin signaling molecules; 2—the effects of eCG on gene and protein expression of INSR, IGF1, GLUT4 and NFKB1A in bovine luteal cells; and 3—the effect of stimulatory and superovulatory treatments on gene and protein expression of ANG, ANGPT1, NOS2, ADM, PRSS2, MMP9 and PLAU. Serum insulin did not differ among groups (P = 0.96). However, serum somatomedin C levels were higher in both stimulated and superovulated groups compared to the control (P = 0.01). In stimulated cows, lower expression of INSR mRNA and higher expression of NFKB1A mRNA and IGF1 protein were observed. In superovulated cows, lower INSR mRNA expression, but higher INSR protein expression and higher IGF1, IGFR1 and NFKB1A gene and protein expression were observed. Expression of angiogenesis and cellular modelling pathway-related factors were as follows: ANGPT1 and PLAU protein expression were higher and MMP9 gene and protein expression were lower in stimulated animals. In superovulated cows, ANGPT1 mRNA expression was higher and ANG mRNA expression was lower. PRSS2 gene and protein expression were lower in both stimulated and superovulated animals related to the control. In vitro, eCG stimulated luteal cells P4 production as well as INSR and GLUT4 protein expression. In summary, our results suggest that superovulatory treatment induced ovarian proliferative changes accompanied by increased expression of genes providing the CL more energy substrate, whereas stimulatory treatment increased lipogenic activity, angiogenesis and plasticity of the extracellular matrix (ECM).

Efficacy of induction of luteolysis in superovulated cows is dependent on time of prostaglandin F2alpha analog treatment: effects on plasma progesterone and luteinizing hormone profiles

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF–induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses.

Preliminary study of factors affecting the superovulatory response of high producing dairy cows superstimulated regardless of the stage of estrous cycle in Egypt

This work was conducted as a first time commercial production of embryos from lactating Holstein and Brown Swiss cows using multiple ovulation embryo transfer (MOET) technology in Egypt. We studied factors affecting the superovulatory response (SR) in superovulated cows and effects of propylene glycol (PG) on embryo quality. Daily milk production at flushing had significantly negative effects on SR and embryo yields in superovulated cows. In addition, Brown Swiss cows had better SR than Holstein cows. Moreover, cows having more than 3 parities yielded better response, compared to cows in the first three parities. However, factors such as body weight at flushing, body condition score (BCS) at flushing and days in milk (DIM) at flushing did not have any association with SR in cows. In addition, drenching of PG prior to and during the superovulatory treatment improved SR (Right CL number, P < 0.05; Left CL number, P < 0.05), total embryos per flush (P < 0.05), first grade embryos per flush (P < 0.01) and tended to improve transferable embryos (P = 0.13) and second grade embryos per flush (P = 0.11). However, it tended to increase the number of degenerated embryos per flush (0.06). In conclusion, commercial production of embryos from lactating Holstein and Brown Swiss cows regardless of stage of estrous cycle by MOET proved successful under Egyptian conditions. Milk yield was negatively associated with SR and embryo yields in superovulated cows. Moreover, drenching of PG prior to and during the superovulatory treatment improved SR and embryo yields.

Treatment with eCG Decreases the Vascular Density and Increases the Glandular Density of the Bovine Uterus

The uterus plays an essential role in mammalian reproduction and is a target of several hormonal protocols used to improve fertility in cattle. Many studies highlighted the importance of eCG treatment following fixed-time artificial insemination in improving follicular growth, ovulation and pregnancy rates in cattle. Moreover, eCG has been implicated in angiogenesis, leading to important changes in uterine blood flow and vascularisation. However, there is still a lack of information regarding the specific alterations induced by eCG upon glandular and vascular characteristics of bovine uterus. To investigate the influence of eCG on: uterine thickness and area; uterine artery diameter and area; uterine vascular and gland density; and the expression of the VEGFA-system, the uteri of crossbred beef cows were collected. All cows were submitted to follicular wave emergence synchronization. On day four of protocol, cows submitted to superovulation (n=6) received 2000IU eCG, on day eight, after expected follicular deviation, cows submitted to stimulatory treatment (n=5) received 400IU eCG. Control cows (n=5) did not receive eCG. On day five po cows were subjected to ultrassonographic evaluation and slaughtered for uterine tissue sampling on day six po. Uterine vessels and glands were quantified by the counting point stereological method. The VEGFA-system was localized in different cellular types, showing no qualitative or quantitative differences in the site of expression or the intensity of the positive signal among the groups. Vascular density was decreased in the endometrium of stimulated and myometrium of superovulated cows compared with the control ones, which showed higher vascular density in the myometrium and endometrium of the ipsilateral uterine horn. The uterine gland density was higher in superovulated compared with stimulated and control cows. Thus, we can infer that stimulatory or superovulatory treatments with eCG influence the vascular density in the endometrium and myometrium in cattle.

Effects of deep-horn AI on fertilization and embryo production in superovulated cows and heifers

The primary objective of this study was to determine the effect of site of semen deposition on fertilization rate and embryo quality in superovulated cows. The hypothesis was that deposition of semen into the uterine horns would increase the fertilization rate compared with deposition of semen into the uterine body. The secondary objective was to evaluate the effect of uterine environment on fertilization rate and embryo quality. It was hypothesized that subclinical endometritis at the onset of superstimulation would decrease the fertilization rates and embryo quality. In experiment 1, 17 superovulated heifers were randomly assigned to receive artificial insemination (AI) into the uterine body or uterine horns. The total number of fertilized structures and fertilization rate from superovulated heifers was increased (P = 0.04 and P = 0.02, respectively) when semen was deposited into the uterine horns compared with the uterine body. Other embryo characteristics did not differ based on the site of semen deposition. In experiment 2, 14 lactating dairy cows were superovulated twice and were randomly assigned to receive AI into the uterine body or deep into the uterine horns using a crossover design. Neither fertilization rate nor any other embryo characteristics were improved when semen was placed deep into the uterine horns compared with the uterine body. In experiment 3, 72 superovulated lactating dairy cows were randomly assigned to receive AI into the uterine body or uterine horns. Before initiation of superstimulatory treatments, an endometrial cytology sample was collected from each cow. Ova/embryos were collected by a nonsurgical technique at 70 ± 3 days in milk. Similar to experiment 2, neither fertilization rate nor any other embryo characteristics differed based on the site of semen deposition in experiment 3. The percentage of cows with subclinical endometritis did not differ between treatments. Interestingly, there was a tendency (P = 0.09) for a reduction in embryo recovery rate and a reduction (P = 0.01) in the fertilization rate for cows with subclinical endometritis. In conclusion, deposition of semen into the uterine horns rather than into the uterine body did not improve the fertilization rate or embryo quality in superovulated cows. Subclinical endometritis decreased the fertilization rate in superovulated cows.

Embryo production with sex-sorted semen in superovulated dairy heifers and cows

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15–hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30–45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8–14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.

Embryos produced from fertilization with bovine viral diarrhea virus (BVDV)-infected semen and the risk of disease transmission to embryo transfer (ET) recipients and offspring

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems in cattle. For that reason, several countries have eradicated the disease, and some others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for artificial insemination (AI), there is no evidence to indicate whether the resulting embryos, when used for embryo transfer, can lead to the transmission of BVDV to recipients or offspring. For this experiment, semen from a bull persistently infected with BVDV (105 50% tissue culture infective doses/mL NY strain) was used for insemination (two times at estrus) of BVDV-seronegative, superovulated cows (N = 35). Embryos were collected 7 days after insemination and subsequently were washed according to the International Embryo Transfer Society recommendations or left unwashed. Out of 302 collected oocytes and embryos, 173 (57%) were fertilized and the remaining 129 (43%) had degenerated. Infectious BVDV was detected in 24% (17/71) of unwashed and 10% (8/77) of washed embryos, and in all (N = 11) follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. After transfer of 39 washed embryos to 27 BVDV-seronegative recipients, 12 (44%) cows became pregnant and 17 calves free of BVDV and BVDV antibodies, including five sets of twins, were born. After embryo transfer, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In conclusion, results herein suggest that BVDV can be transmitted by AI resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to International Embryo Transfer Society and the World Organization for Animal Health guidelines do not cause BVDV transmission to recipients or their offspring.

In vivo embryo production in cows superovulated 1 or 2 days after ovum pick-up

The present study evaluated superovulatory responses and in vivo embryo production in cows treated with FSH starting 1 or 2 days after ovum pick-up (OPU). Thirty-three non-lactating Nelore cows were subjected to aspiration of all follicles ≥3mm for OPU. After OPU, cows were randomly divided into two groups in which the follicle superstimulatory treatments with FSH started 1 or 2 days after OPU (Groups D1 and D2, respectively). Data are presented as the least squares mean±s.e.m. The number of follicles ≥3mm before OPU was similar between groups (~34); however, cows in Group D2 had more follicles ≥3mm on the first day of FSH (15.2±2.3 vs 7.6±1.7; P=0.04) and a higher ratio of the number of follicles at first FSH/number of follicles before OPU (0.41±0.04 vs 0.24±0.02; P=0.01). In addition, Group D2 cows had a greater superovulatory response than did cows in Group D1 (18.9±2.8 vs 9.1±1.9 corpora lutea, respectively; P<0.03). However, there was no difference in the total number of recovered ova and embryos from cows in Groups D2 and D1 (5.1±1.4 vs 4.9±1.3, respectively; P>0.10). Nevertheless Group D2 cows had more freezable embryos than Group D1 cows (3.2±1.1 vs 1.3±0.5, respectively; P<0.05). Cows from Group D2 had a much higher proportion (P<0.001) of follicles ≥8mm compared with follicles ≥6mm and <8mm at the time of the last treatment with FSH. In conclusion, to obtain a greater production of viable embryos in superovulated cows after OPU, it is recommended to wait at least 2 days before starting FSH treatment.

Equine chorionic gonadotropin alters luteal cell morphologic features related to progesterone synthesis

Exogenous eCG for stimulation of a single dominant follicle or for superovulation are common strategies to improve reproductive efficiency by increasing pregnancy rates and embryo production, respectively. Morphofunctional changes in the CL of eCG-treated cattle include increases in CL volume and plasma progesterone concentrations. Therefore, we tested the hypothesis that eCG alters the content of luteal cells and mitochondria related to hormone production. Twelve crossbred beef cows were synchronized and then allocated into three groups (four cows per group) and received no further treatment (control) or were given eCG either before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Six days after ovulation, cows were slaughtered and CL collected for morphohistologic and ultrastructural analysis. Mitochondrial volume per CL was highest in superovulated followed by stimulated and then control cows (18,500 ± 2630, 12,300 ± 2640, and 7670 ± 3400 μm3; P < 0.001), and the density of spherical mitochondria and the total number of large luteal cells were increased (P < 0.05) in stimulated cows compared with the other two groups (110.32 ± 14.22, 72.26 ± 8.77, and 70.46 ± 9.58 mitochondria per μm3 and 678 ± 147, 245 ± 199, and 346 ± 38 × 106 cells, respectively. However, the largest diameters of the large luteal cells were increased in superovulated and control cows versus stimulated ones (32.32 ± 0.06, 31.59 ± 0.81, and 29.44 ± 0.77 μm; P < 0.0001). In contrast, the total number of small luteal cells was increased in superovulated cows (1456 ± 268, 492 ± 181, and 822 ± 461 × 106, P < 0.05). In conclusion, there were indications of cellular changes related to increased hormonal production (stimulatory treatment) and increased CL volume (superovulatory treatment).

Global gene expression in the bovine corpus luteum is altered after stimulatory and superovulatory treatments

Equine chorionic gonadotrophin (eCG) has been widely used in superovulation and artificial insemination programmes and usually promotes an increase in corpus luteum (CL) volume and stimulates progesterone production. Therefore, to identify eCG-regulated genes in the bovine CL, the transcriptome was evaluated by microarray analysis and the expression of selected genes was validated by qPCR and western blot. Eighteen Nelore crossbred cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronised using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG at device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea were collected 7 days after gonadotrophin-releasing hormone administration. Overall, 242 transcripts were upregulated and 111 transcripts were downregulated in stimulated cows (P ≤ 0.05) and 111 were upregulated and 113 downregulated in superovulated cows compared to the control animals (1.5-fold, P ≤ 0.05). Among the differentially expressed genes, many were involved in lipid biosynthesis and progesterone production, such as PPARG, STAR, prolactin receptors and follistatin. In conclusion, eCG modulates gene expression differently depending on the treatment, i.e. stimulatory or superovulatory. Our data contribute to the understanding of the pathways involved in increased progesterone levels observed after eCG treatment.

Effects of polymorphonuclear neutrophile infiltration into the endometrial environment on embryonic development in superovulated cows

Recent studies on bovine uterine disorders have demonstrated that endometrial infiltration with polymorphonuclear neutrophils (PMN) in the postpartum period or at the time of breeding negatively affects reproductive performance. The objective of the present study was therefore to analyze the effect of endometrial PMN infiltration on superovulation outcome. Cows were synchronized and superovulated receiving a total of three artificial inseminations within 24 h. Endometrial cytologic samples were collected by cytobrush technique at first artificial inseminations (AI) (d −1) and before embryo flush (d 7). Embryos were recovered by uterus flushing at Day 7 and evaluated for total cell number and apoptotic cell index. A total of 425 embryos were flushed out of 48 superovulated cows. The PMN dynamics from first AI to flushing had a significant effect on flushing outcome. Significant differences in terms of number of palpable corpora lutea (14.1 vs 7.2) and transferable embryos (8.8 vs 1.9) were found between cows with PMN proportions increasing from zero (0%) at AI to positive proportions (> 0%) at flushing (group PMNZP) and cows with higher endometrial PMN proportions decreasing to lower but still positive proportions from AI to flushing (group PMNHL). Moreover, cows classified to PMN class zero at first AI flushed a significant higher number of total embryos (10.3 vs 6.9) and transferable embryos (6.8 vs 3.7) compared to cows of PMN class positive at first AI (P > 0.05) in our study. Considering a significant interaction effect between PMN class at first AI and flush (P < 0.05), PMN class at first AI (d −1) correlated significantly with number of total flushed and transferable embryos only in combination with a positive PMN class at flush (d 7). Likewise, PMN class at flush (d 7) beard a significant effect on total number of flushed embryos only when classified to PMN class zero at first AI. Collectively, the present work is the first study that demonstrated a significant relationship between endometrial PMN infiltration at first AI as well as PMN dynamic from first AI to time of flush and superovulation outcome.

Behavioral Characteristics of Bos indicus Cattle after a Superovulatory Treatment Compared to Cows Synchronized for Estrus

The intensity and duration of sexual behavior in Bos indicus was assessed through the continuous observation of sexual receptivity. Two groups of cows were formed: only synchronized (n = 50) and other group further superovulated (n = 20). An intravaginal implant that released progesterone over 9 d was used. After removing the implant, 25 mg of PGF2α was administered. In the superovulated group, the administration of 280 mg (Follicle stimulant hormone) FSH-P1 per cow with a decreasing dosage over 4 d was utilized. In both groups, behavioral observations began at the moment of implant removal. Sexual behavior was analyzed using a Kruskal-Wallis test to compare the mean of hours in estrus, effective mountings and number of mounts/hour during estrus. A nonparametric survival analysis was performed using the time in two ways: i) when an event happened it was placed in a 24 h time- frame and, ii) the time of observation in continuous form (96 h) assessing the difference between curves by the log rank test Chi-square. The only significant difference was the number of mounts/h during receptivity (p<0.05). In the superovulated group three periods of sexual activity during the day were identified, with these events being of greater frequency and duration than the synchronized group (p = 0.02); besides, the superovulated group began estrus before the synchronized group (p = 0.0035) when using the total period. In a simulation study, when the number of observations went from two (06:00-18:00) to three periods (06:00, 12:00 and 18:00) cows detected accurately (<6 h after the onset) increased more than 20%. The results show that superovulated cows presented greater intensity

323 EFFICIENCY OF CLOPROSTENOL-INDUCED LUTEOLYSIS IN SUPEROVULATED COWS

The induction of multiple ovulations is a key procedure for in vivo embryo production. Many corpora lutea (CL) are developed, resulting in abnormally high progesterone concentrations. Luteolysis induction by prostaglandin F2α and its analogues is well described in cows bearing one or few, but not multiple, CL, as occurs after superovulation. The objective of the first experiment was to compare the efficacy of a single prostaglandin F2α treatment on inducing luteolysis in embryo donors immediately after flushing (D7, N = 24) or 4 days later (D11, N = 22). Holstein cows were superovulated with 400 IU of FSH following standard procedures. Embryo flushing was performed 7 days after AI, and cows were randomly allocated into 2 groups to receive either a 0.5 mg of sodium cloprostenol IM treatment immediately after flushing (D7 group) or the same treatment 4 days later (D11 group). Occurrence of luteolysis was monitored by plasma progesterone concentrations (P4), measured by radioimmunoassay in blood samples taken at 4-h intervals. There was no difference in P4 before treatment between D7 and D11 groups (28.6 ± 5.2 v. 36.4 ± 7.4 ng mL–1, respectively; P &gt; 0.05). Although cloprostenol caused a remarkable decline in P4 48 h after treatment in both groups (1.8 ± 0.3 and 1.6 ± 0.4 ng mL–1 for D7 and D11 groups, respectively; P &lt; 0.05), P4 continued decreasing in D11 cows thereafter, remaining smaller than 1 ng mL–1 up to 196 h after treatment, whereas in D7 cows, there was no further reduction in P4. Luteolysis (P4 &lt;1 ng mL–1) was observed in all D11 cows, but failed in 11 of 20 (55%) D7 cows, in which P4 increased after the initial cloprostenol-induced decrease. The second experiment compared luteolysis in superovulated v. nonsuperovulated cows. Non-superovulated (control group, CG, N = 8) and superovulated cows (SOV, N = 6) received a single dose of sodium cloprostenol IM (0.5 mg) on day 11 after oestrus. Morphological and functional luteolysis were monitored daily by ovarian ultrasonography and P4 analysis; also, plasma LH was measured in blood samples taken every 20 min for 1 h, during 5 days. Individual CL size was smaller (1.8 ± 0.1 v. 3.5 ± 0.4 cm2) but total luteal tissue was greater (29.8 ± 7.0 v. 3.5 ± 0.4 cm2; P &lt; 0.05) in SOV than in CG. A considerable decrease in P4 occurred in both groups 24 h after treatment (from 51.1 ± 12.9 to 5.1 ± 0.9 ng mL–1 in SOV and from 5.9 ± 0.6 to 1.1 ± 0.1 ng mL–1 in CG); however, SOV cows did not reach P4 values similar to CG until 96 h after treatment (0.9 ± 0.3 v. 0.4 ± 0.2 ng mL–1, respectively; P &gt; 0.05). There was no difference in initial LH values between SOV and CG (1.5 ± 0.1 and 1.5 ± 0.1 ng mL–1; P &gt; 0.05), but the slower decrease in P4 in the SOV group prevented LH from increasing up to 96 h after luteolysis induction, whereas mean LH values increased (P &lt; 0.05) in CG after 24 h. In conclusion, luteolysis failure may occur when cloprostenol is given at the day of flushing (7 days after AI) in superovulated cows. In addition, luteolysis induction on day 11 after SOV is efficient, but the initial high progesterone concentration results in a slower rate of P4 decrease to basal levels. The authors acknowledge CNPq and FAPEMIG Project CVZ AQP 01654/09.