Cyclic Cows Research Articles

Comparative Studies on Blood Biochemical Profile of Anestrus and Normal Cyclic Jersey Cross-Bred Cows

Comparative Studies on Blood Biochemical Profile of Anestrus and Normal Cyclic Jersey Cross-Bred Cows

New Approach for Improvement of the First Insemination Conception rate in Ovsynch Treated Holstein Dairy Cows by Use of Estradiol Benzoate and Antioxidants

The aim of the current study was conducted to improve the first insemination conception rate in Holstein dairy cows by the use of estradiol benzoate and antioxidants in conjunction with ovsynch program. A total number of 160 cyclic dairy cows 55-67 day in milk, 4-10 years old, 2nd to 5th lactations were divided into 4 groups: G1 (n=40) treated with ovsynch program (control group). G2 (n=40) injected with 1mg estradiol benzoate i.m at the time of estrumate injection in ovsynch protocol, G3 (n=40) injected with 15ml of vitesel i.m at the time of estrumate injection in ovsynch protocol, G4 (n=40) injected with 1mg of estradiol benzoate plus 15ml vitesel i.m at the time of estrumate injection in ovsynch protocol. The expression ad intensity of estrus signs were studied using behavioral and physiological changes. The conception rate after first insemination examined using transrectal ultrasonography on the 30th day from insemination and pregnancy rate was confirmed rectally after 60 days from insemination. The obtained results showed that total cows expressed estrus in G2 (ovsynch plus estradiol benzoate) and G4 (ovsynch plus estradiol benzoate and E-selenium) had highly significantly incidence (87.5% and 95.0%) compared with the cows of G1 (ovsynch group) and G3 (ovsynch plus E-selenium) 52.5% and 60%) P < 0.0001. The G2 and G4 had significant incidence of intense estrus signs (82.9% and 84.2%) compared with cows of G1 and G3 (57.1% and 62.5%) p = 0.040. also, it was noted that the G2 and G4 had highly significant incidence of first insemination conception (75.5% and 80%) versus (32.5% and 40%) in cows of G1 and G3 P < 0.0001. the pregnancy rate in G2 and G4 had highly significant incidence (67.5% and 80%) versus (30% and 40%) in cows of G1 and G3 P < 0.0001 as shown in table 2. Moreover, all cows showed intense estrus signs had 100% first insemination conception rate compared with 6.7% in weak estrus signs as in (table 3 and figure 1). It can be concluded that the beneficial effects of injection of estradiol benzoate and antioxidant with ovsynch program improve the reproductive efficiency through intensified signs of estrus and increase secretary activity of the tubular tract. These drugs improve the first insemination conception, pregnancy rates and enhances fertility in postpartum Holstein dairy cows.

Endometrial mRNA expression of selected pro-inflammatory factors and mucins in repeat breeder cows with and without subclinical endometritis

Repeat breeder cows (RBC) are defined as cyclic cows without clinical abnormalities that fail to conceive after at least three subsequent inseminations. Previous studies have elucidated cellular defence mechanisms in the bovine uterus but detailed information on inflammatory events of endometrial cells in RBC is still lacking. Thus, the objective of this study was to analyse endometrial mRNA expression of selected transcripts associated with uterine inflammatory processes. Cytobrush samples from 91 RBC and 11 synchronised heifers with no history of gynaecological abnormalities (controls, CON) were collected. The proportion of polymorphonuclear neutrophils in these samples was used for the diagnosis of subclinical endometritis (SE). Ultrasonography and progesterone blood concentrations were used to determine ovarian activity and the stage of the oestrous cycle. Total RNA was isolated from the cytobrush samples and subjected to reverse transcription-quantitative PCR for interleukins (IL) 1A, IL1B, IL6, IL8, chemokine CXL ligand (CXCL) 3, CXCL5, prostaglandin-endoperoxide synthase 2 (PTGS2), tracheal antimicrobial peptide (TAP) and mucin (MUC) 4, MUC5, MUC6, MUC12 and MUC16. CXCL3 mRNA was higher (2-fold) and PTGS2 mRNA lower (6-fold) expressed in RBC compared with CON (P < 0.05). After subdivision of RBC in animals with (RBC-SE) and without SE (RBC-noSE), these differences remained significant between RBC-noSE and CON. Higher mRNA abundances of IL1A and IL1B were found in RBC-SE compared with RBC-noSE (3- and 4-fold; P < 0.05). No differences in the mRNA expression of IL6, IL8, CXCL5 and TAP were observed between RBC-SE, RBC-noSE and CON. MUC4 and MUC12 mRNA was more highly expressed in RBC than in CON (P < 0.05). In RBC-noSE, a 5- and 14-fold higher MUC4 and MUC12 mRNA expression was noticed compared with CON (P < 0.05). A significantly lower mRNA expression of MUC5 and MUC16 (7- and 4-fold) was detected in RBC in the luteal phase compared with RBC in the follicular phase, whereas such a down-regulation was not observed for MUC4 and MUC12. In conclusion, we demonstrated different PTGS2 and CXCL3 mRNA expression between RBC and control heifers, which might be related to subfertility in RBC. Further studies are required to confirm that an unregulated MUC4 and MUC12 mRNA expression may contribute to subfertility of RBC. These findings provide a valid basis for further research on regulatory mechanisms of mRNA expression in subfertile cows.

Embryo Mortality Around the Period of Maintenance of the Corpus Luteum Causes Alterations to the Ovarian Function of Lactating Dairy Cows.

Objectives were to identify cows with embryo mortality (EM) around the period of corpus luteum maintenance by interferon tau (IFNT) and to characterize ovarian function in cows that underwent EM. Lactating Holstein cows received artificial insemination (AI) (Day = 0) with semen or extender only. From Day 14 to 42 transrectal ultrasonography was performed daily to monitor ovarian dynamics and uterine contents whereas blood was collected every 48 h to determine ISG15 and MX2 mRNA abundance in blood mononuclear cells (Day 14 to 22 only) and determination of hormone concentrations. Cows were classified in the following reproductive status groups: cyclic (inseminated with extender; n = 15), pregnant (embryo present on Day 42; n = 23), no embryo (n = 23), and EM (n = 14). EM was defined as the presence of an embryo based on interferon-stimulated genes (ISG) mRNA abundance and concentrations of pregnancy-specific protein B (PSPB) above specific cutoff points but no embryo visualized by ultrasonography. Within the EM group, early EM (up to Day 22) was when ISG fold changes were above specific cutoff points from Day 18 to 22 and PSPB below 0.7 ng/ml on and after Day 24, whereas late EM (after Day 22) was when PSPB was above 0.7 ng/ml on or after Day 24 regardless of ISG expression. This experiment provided evidence that the combination of ISG expression patterns and PSPB concentrations is a reasonable method to determine EM around the period of corpus luteum maintenance by IFNT because cows with evidence of EM had patterns of ISG expression more similar to pregnant than cyclic cows or cows with no embryo. Within the EM group, only cows with late EM had delayed luteal regression and longer interovulatory intervals. No major alterations in follicular function were observed after the onset of luteolysis. Our results suggest that embryo development needs to continue beyond 22 days after AI to effectively prevent luteolysis and extend the luteal phase.

Regulation of the bovine oviductal fluid proteome.

Our objective was to investigate the regulation of the proteome in the bovine oviductal fluid according to the stage of the oestrous cycle, to the side relative to ovulation and to local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected at four stages of the oestrous cycle: pre-ovulatory (Pre-ov), post-ovulatory (Post-ov), and mid- and late luteal phases from adult cyclic cows (18-25 cows/stage). The proteomes were assessed by nanoLC-MS/MS and quantified by label-free method. Totally, 482 proteins were identified including a limited number of proteins specific to one stage or one side. Proportions of differentially abundant proteins fluctuated from 10 to 24% between sides at one stage and from 4 to 20% among stages in a given side of ovulation. In oviductal fluids ipsilateral to ovulation, Annexin A1 was the most abundant protein at Pre-ov compared with Post-ov while numerous heat shock proteins were more abundant at Post-ov compared with Pre-ov. Among differentially abundant proteins, seven tended to be correlated with intra-oviductal concentrations of progesterone. A wide range of biological processes was evidenced for differentially abundant proteins, of which metabolic and cellular processes were predominant. This work identifies numerous new candidate proteins potentially interacting with the oocyte, spermatozoa and embryo to modulate fertilization and early embryo development.

Exposure of Lactating Dairy Cows to Acute Pre-Ovulatory Heat Stress Affects Granulosa Cell-Specific Gene Expression Profiles in Dominant Follicles.

High environmental temperatures induce detrimental effects on various reproductive processes in cattle. According to the predicted global warming the number of days with unfavorable ambient temperatures will further increase. The objective of this study was to investigate effects of acute heat stress during the late pre-ovulatory phase on morphological, physiological and molecular parameters of dominant follicles in cycling cows during lactation. Eight German Holstein cows in established lactation were exposed to heat stress (28°C) or thermoneutral conditions (15°C) with pair-feeding for four days. After hormonal heat induction growth of the respective dominant follicles was monitored by ultrasonography for two days, then an ovulatory GnRH dose was given and follicular steroid hormones and granulosa cell-specific gene expression profiles were determined 23 hrs thereafter. The data showed that the pre-ovulatory growth of dominant follicles and the estradiol, but not the progesterone concentrations tended to be slightly affected. mRNA microarray and hierarchical cluster analysis revealed distinct expression profiles in granulosa cells derived from heat stressed compared to pair-fed animals. Among the 255 affected genes heatstress-, stress- or apoptosis associated genes were not present. But instead, we found up-regulation of genes essentially involved in G-protein coupled signaling pathways, extracellular matrix composition, and several members of the solute carrier family as well as up-regulation of FST encoding follistatin. In summary, the data of the present study show that acute pre-ovulatory heat stress can specifically alter gene expression profiles in granulosa cells, however without inducing stress related genes and pathways and suggestively can impair follicular growth due to affecting the activin-inhibin-follistatin system.

Effects of breed, management system, milk yield and body weight on onset of postpartum ovarian cyclicity in cows

To determine the factors affecting onset of postpartum ovarian cyclicity (PPOC) milk progesterone concentrations were measured in 84 cows. About 30% showed cyclicity within 120 days of parturition. Intensively managed cows started cyclicity earlier (P&gt;0.05) than extensively managed animals. Holstein-Friesian crossbred cows started ovarian cyclicity earlier (80.2 ± 17.5 days) with higher proportion (37.9%) than local zebu cows (84.8 ± 21.0 days) and about 13.3%, respectively (P&gt;0.05). The cows yielding 4-12 kg milk/day required 73.5 ± 10.0 days to start ovarian function earlier than those producing 1-2 kg/day (84.8 ± 21.0 days) (P&gt;0.05). Cows with lower (&lt;200 kg) and higher (&gt;300 kg) body weights started ovarian activity by 81.6 ± 16.6 and 76.3 ± 17.4 days, respectively (P&gt;0.05). Cows with lower body weight showed PPOC at lower rate (5.2%) than those of medium (38.3%) and higher body weight (33.3%) (P&lt;0.05). The delayed onset of PPOC prolonged the calving interval.Bangl. vet. 2015. Vol. 32, No. 1, 27-34

Effect of estrus synchronization on plasma progesterone profile and fertility response in postpartum suckled anestrous Kankrej cows

The study was designed at evaluating early postpartum ovarian activity through plasma progesterone profile at 10 days interval up to 90 days, followed by induction of estrus in postpartum anestrous suckled Kankrej cows (n=18) using Ovsynch, CIDR and Ovsynch + CIDR treatment protocols with FTAI (6 cows in each protocol), keeping six normal cyclic cows as control. The plasma P4 profile (ng/ml) varied in the range of 0.15±0.03 to 1.70±0.80 in treatment groups and 0.62 ±0.19 to 4.09±1.60 in control group up to 90 days postpartum. The mean P4 concentration on the day of calving was low (&lt;1 ng/ml) in all the groups, which gradually increased to reach peak levels by day 40 in cyclic control (4.09±1.60 ng/ml), Ovsynch (1.70±0.80 ng/ml) and CIDR (1.68±0.47 ng/ml) and by day 20 in Ovsynch + CIDR (1.09±0.23 ng/ml) groups. Most of the cows turned into subestrous or anestrous condition between days 40 and 90 postpartum. The estrus induction response in Ovsynch, CIDR and Ovsynch + CIDR groups initiated at day 90-92 postpartum was 66.66, 83.33 and 50.00 per cent, respectively. The conception rates at induced estrus were 16.66, 33.33 and 16.66 per cent, and overall of 3 cycles 33.33 (2/6), 50.00 (3/3) and 33.33 (2/6) per cent, respectively. In normal cyclic control group, the conception rates at first cycle and overall of 3 cycles were 33.33 and 50.00 per cent. The mean plasma P4 (ng/ml) concentrations were significantly (P&lt;0.05) higher on day 7 in Ovsynch (1.41 ±0.10), CIDR (4.92±0.83) and Ovsynch + CIDR (3.87±0.84) protocols as compared to their corresponding values on day 0 and day 9/10 (AI). The mean values of plasma P4 from days 20 to 40 post-AI were higher in conceived cows than in non-conceived cows of CIDR and Ovsynch + CIDR groups, compared to the animals in Ovsynch and normal cyclic groups. It is opined that the use of different protocols, mainly CIDR, may serve as an effective tool for induction and synchronization of estrus and improvement of conception rate in postpartum anestrous suckled Kankrej cows.

Effect of Serum Calcium, Magnesium, and Phosphorus Levels in Cyclic and Postpartum Anestrus Jersey Cross Cows

Effect of Serum Calcium, Magnesium, and Phosphorus Levels in Cyclic and Postpartum Anestrus Jersey Cross Cows

Metabolic profiles of high-yielding dairy cows with ovarian cysts formation

Background: Ovarian cysts are among the diseases which cause reproductive failure and economic losses in dairy herds. High yelding dairy cattles are suseptable to reproductive failure caused by ovarian cysts, as a result of their exposure to stressful coditions during lactation. OBJECTIVES: This study aimed to monitor metabolic profile tests in dairy cows with ovarian cysts in comparison with cycling cows. METHODS: Forty high-yelding Holstein dairy cows were enrolled for this study (20 cows with ovarian cysts and 20 cyclic cows). Seven weeks after parturation, 40 cows without retained placenta, including healthy ones were selected. Ovarian cysts were detected as follicular-like structures, >20 mm in diameter, persisting for at least 7 days, without corpus luteum and were monitored by ultrasound examination. Blood samples were collected from the jugular vein and delivered to the laboratory for measurement of calcium, magnesium, phosphorus, sodium, potassium, beta-hydroxy butyric acid, non esterified fatty acids (NEFA), glucose, cortisol, insulin and BUN. RESULTS: In this study, when serum calcium, phosphorus, sodium, potassium, magnesium and glucose of cows with ovarian cysts were compared with cows that had normal ovarian status, there was no significant difference. BUN, beta-hydroxy butyrate, NEFA and cortisol of cows with ovarian follicular cysts as compared to the cows with normal ovaries, showed a significant increase. Serum insulin values decreased significantly in ovarian follicular cystic cows than in cyclic cows. CONCLUSIONS: Increasing blood cortisol and decreasing blood insulin may play a major role in the formation of ovarian cyst and any detectable change in NEFA, BHBA and BUN.

Effects of induced endometritis on uterine blood flow in cows as evaluated by transrectal Doppler sonography.

This study was conducted to evaluate the effects of induced endometritis on uterine blood flow in cows. Transrectal Doppler sonography was performed on uterine arteries of six cyclic cows before and for 4 days after inducing acute endometritis by intrauterine infusion of 720 mg of policresulen, and for 4 days of the following estrous cycle. Time-averaged maximum velocity (TAMV) increased (p < 0.001) and pulsatility index (PI) decreased (p < 0.0001) within 1 h of policresulen administration, and did not change (p > 0.05) in the next 4 days of the same cycle. TAMV and PI values in the subsequent cycle did not differ (p > 0.05) from the values measured before infusion and showed no changes (p > 0.05) within the cycle. Blood flow parameters were not related (p > 0.05) to plasma concentrations of progesterone and estrogen. All cows showed an acute endometritis determined by histopathological findings of biopsy samples taken 1 day after infusion and fibrotic endometrial alterations detected in the subsequent cycle. No relationships were observed between fibrotic changes of the endometrium and uterine blood flow during either cycle. In conclusion, acute inflammation is accompanied by a rise in uterine blood flow, but fibrotic alterations do not seem to be related to Doppler sonographic findings.

Aspects and mechanisms of low fertility in anovulatory dairy cows

Postpartum anovulation is a natural process that is observed in most mammals, including women. In lactating dairy cows, the interval from calving to first ovulation typically averages 4 to 5 weeks, but a substantial proportion of cows have not resumed estrous cyclicity by 60 days postpartum. Extended delay in resumption of first postpartum ovulation is known to exert long-lasting detrimental effects on fertility in dairy cows including the lack of spontaneous estrus and subsequent timely insemination postpartum, but when anovular cows have the estrous cycle synchronized for artificial insemination (AI), still pregnancy per AI is reduced and the risk of pregnancy loss increased. Many risk factors exist for extended postpartum anovulatory periods such as negative nutrient balance and diseases, and these risk factors are also known to depress fertility by themselves. A key feature in anovular cows when inseminated is that they develop the ovulatory follicle under subluteal or low concentrations of progesterone. Progesterone from the corpus luteum is pivotal for follicle development, oocyte competence, embryo growth, and endometrial function; however, many of these effects exerted by progesterone are mediated either by secretion of gonadotropins influencing follicular function and oocyte competence or by endometrial histotroph secretion influencing embryo/conceptus growth and developmental biology. Sub-optimal concentrations of progesterone during follicle growth in anovular cows affect morula and early blastocyst quality, alter conceptus gene expression and affect endometrial function increasing the synthesis of prostaglandin F2α. When anovular cows receive sufficient supplemental progesterone during the antral stages of development of the ovulatory follicle, then pregnancy per AI is reestablished and resembled that of estrous cyclic cows that developed the follicle during diestrus. Preliminary data suggest that a minimum concentration of progesterone during follicle growth is needed to optimize fertility in anovular cows, at least 2.0 ng/ml.

Evaluation of micromineral status and ovarian hormonal profile and their interrelation in normal cyclings and repeat breeding cows

Blood samples from 12 normal cycling and 12 repeat breeding crossbred cows were collected aseptically on different phages of oestrous cycle to elucidate the possible relationship between the trace elements and hormone profile of ovary in normal cycling and repeat breeding cross bred cows. Different trace elements namely Fe, Cu, Zn and Mn were estimated using Atomic Absorption Spectrophometer (AAS) and serum oestradiol 17-β and progesterone were estimated using coatA-count ® RIA kit. Fe, Cu and Mn concentration was observed apparently high and Zn concentration was significantly (Pβ0.05) high in each phase of collection in normal cycling cows from repeat breeder except mid luteal phase. Oestradiol 17-β concentration was significantly (Pβ0.01) high in normal cycling cows in oestrous and early luteal phase, while no significant variation was observed for progesterone concentration between the same phase of the two groups. A positive significant (Pβ0.01) correlation was observed between Fe-Zn and Cu-Zn (Pβ0.05) and oestradiol 17-β (Pβ0.05) in normal cycling cows . In repeat breeding cows Fe and oestradiol 17-β and Mn and progresterone was positively significant (Pβ0.01).

108 FACTORS AFFECTING PREGNANCY RATES IN RECIPIENTS RECEIVING IN VITRO-PRODUCED EMBRYOS BY FIXED TIME EMBRYO TRANSFER

A retrospective analysis of factors that affect pregnancy rates from 4214 fresh in vitro-produced (IVP) embryos that were transferred at a fixed-time (FTET) in 20 different farms. Recipients were all cycling cows or heifers that were synchronized with 1 of 3 treatments: 1) treatments with progesterone (P4) devices and 2 mg of oestradiol benzoate (EB) on Day 0 (day of insertion) and 24 h after device removal (Day 8); 2) treatments with P4 devices and EB on Day 0, but with 0.5 mg of oestradiol cypionate (ECP) at device removal (Day 8); or 3) treatments with P4 devices and GnRH on Day 0 and a second GnRH 60 h after device removal (Day 5). Cows in all treatment groups also received 500 µg of cloprostenol (prostaglandin F2α) at the time of P4 device removal and 400 IU of eCG either at device removal or 3 days before device removal. All embryos were transferred 7 or 8 days after the expected time of oestrus (24 h after EB, 48 h after ECP or at the time of the second GNRH for each synchronization treatment, respectively). On the day of embryo transfer, recipients were examined by ultrasonography and those with corpus luteum &gt;14 mm in diameter received a fresh, IVP embryo in the uterine horn ipsilateral to the corpus luteum. Pregnancy rates were determined by ultrasonography 35 days after FTET. Data were analysed by logistic regression. Independent variables were classified into the following three categories. 1) Factors related to the recipient and the environment; there were no significant differences in pregnancy rates for corpus luteum diameter (≥14 and &lt;16 mm, ≥16 and &lt;18 mm, or ≥18 mm; P = 0.46), number of corpus luteum (1 or ≥2; P = 0.26), and category of recipient (cow or heifer; P = 0.21). However, there were significant effects of farm (P = 0.01) and body condition score (BCS; P = 0.01). Cows with BCS ≥4.5 (1 to 5 scale) resulted in lower pregnancy rates (4/20, 20.0%) than those with BCS 2 (74/225, 32.9%), 2.5 (502/1434, 35.0%), 3 (570/1467, 38.9%), 3.5 (193/532, 36.3%), and 4 (44/118, 37.3%). 2) Factors related to the synchronization treatment; there were no significant differences between recipients receiving eCG at device removal (84/209, 40.2%) or 3 days before device removal (874/2291, 38.1%; P = 0.35). However, recipients synchronized with P4 devices and ECP had higher (P = 0.01) pregnancy rates (232/483, 48.0%) than those treated with EB (679/1888, 36.0%) or gonadotropin-releasing hormone (47/129, 36.4%). 3) Factors related to the embryo transfer technique; day of the recipient’s oestrous cycle (P = 0.36), stage of embryo transferred (IETS stages 6 or 7; P = 0.62), and operator (P = 0.57) did not affect pregnancy rates. However transfers made in the anterior third of the uterine horn resulted in higher (649/1545, 42.0%) pregnancy rates than those in the mid-third (845/2511, 33.6%) or in the distal third (6/35, 17.1%; P = 0.01). It was concluded that factors related to the recipient and the environment (farm and BCS), the synchronization treatment (ECP), and the embryo transfer technique (site of deposition) affect pregnancy rates in recipients of embryos produced in vitro and transferred at a fixed time.

134 FOLLICULAR DYNAMICS OF Gyr AND HOLSTEIN OOCYTE DONORS KEPT UNDER TROPICAL CONDITIONS

Bovine in vitro production is highly relevant for dairy systems in Brazil, and the main breeds used as oocyte donors are Gyr (G) and Holstein (H). This study aimed to evaluate the ovarian follicular dynamics of G and H oocyte donors kept under tropical conditions to detect differences that could guide improvement of follicular wave synchronization protocols for ovum pickup. Fourteen cyclic cows (6 H and 8 G), assessed twice each (after a 14-day interval), had their oestrus cycle synchronized by the use of 1.0 g of progesterone via intravaginal device (Ourofino, Brazil) and administration of 2 mg of oestradiol benzoate (EB; Day 0). Withdrawal of progesterone device was followed by 7.6 mg of cloprostenol administration (Day 7); EB (1 mg) was administered after 24 h (Day 8, 0 h), and the animals were evaluated every 12 h by ultrasound for 6 days (0–132 h). All evaluations are reported regarding EB administration (0 h). Videos from each ovary were stored and processed using the ImageJ software (http://rsb.info.nih.gov/ij), by measuring the diameter of each visualised follicle. All procedures were approved by local ethics committee. Ovulation time (G = 42.0 ± 8.3; H = 42.5 ± 6.2), ovulatory follicle (F1) diameter (G = 11.5 ± 1.8; H = 12.4 ± 2.0), and F2 diameter (G = 7.2 ± 1.9; H = 7.4 ± 2.7) did not differ (P &gt; 0.05) between breeds. Growth rate (mm day–1) after ovulation was similar (P &gt; 0.05) between breeds for each follicle (F1 = G: 0.6 ± 0.2, H: 0.8 ± 0.1; F2 = G: 0.5 ± 0.1 H: 0.4 ± 0.1, F3 = G: 0.2 ± 0.1, H: 0.3 ± 0.1). In H group, the F1 growth rate was higher (P &lt; 0.05) than F2 and F3, but there was no difference (P &gt; 0.05) in G group. Follicle deviation was identified 120 h after EB in the G group (~78 h after ovulation) and 132 h in the H group (~90 h after ovulation), and at that time F1, F2, and F3 follicle diameters were 8.0 ± 0.3, 6.6 ± 0.5, and 5.3 ± 0.3 for G (120 h), respectively; 8.8 ± 0.7, 7.2 ± 0.4, and 6.2 ± 0.3 for H (132 h), respectively. There was no difference between the size of F1, F2, and F3 between breeds at any time, except at 132 h, when H F3 was higher (P &lt; 0.05) than G F3. Regarding the follicular population, follicles smaller than 3 mm were more numerous in G animals at all evaluated moments, and differed at 0 (G = 7.1 ± 1.1; H = 2.5 ± 0.5) and 132 h (G = 5.6 ± 0.8; H = 1.5 ± 0.3). Number of follicles between 3 and 8 mm increased in H compared to G at 24 (14.4 ± 1.0), 36 (15.7 ± 1.3), and 132 h (18.3 ± 1.4). Comparing 3- and 8-mm follicles in G between times, an increase (P &lt; 0.05) in number was detected from 36 h onwards, comparedto 0 h (0 h: 9.2 ± 1.0; 36 h: 13.6 ± 1.4). This increase was not significant in H group (0 h: 13.7 ± 1.1; 132 h: 18.3 ± 1.4). The main findings of this study are that the moment of deviation and the population of follicles smaller than 3 mm and between 3 and 8 mm differs from Gyr and Holstein oocyte donors. Those observations suggest ovum pickup is better performed slightly later in Holstein donors than in Gyr, and can contribute to improvement of follicular wave synchronization protocols for each of the breeds in tropical conditions. Study supported by FAPERJ, Embrapa and CNPq.

Comparative efficacy of different estrus synchronization protocols on estrus induction response, fertility and plasma progesterone and biochemical profile in crossbred anestrus cows.

Aim:To evaluate estrus induction response and fertility including plasma progesterone and biochemical profile following use of three standard hormonal protocols in anestrus crossbred cows.Materials and Methods:The study was carried out on 40 true anestrus and 10 normal cyclic cows. 10 anestrus cows each were treated with standard intravaginal controlled internal drug release (CIDR) device, Ovsynch (GPG) protocol, and Norgestomet ear implant with fixed-time artificial insemination (FTAI). 10 anestrus cows were kept as untreated control while 10 cows exhibiting the first estrus within 90 days postpartum without any treatment served as normal cyclic control. Blood samples were obtained from treated cows on day 0, 7, 9 (AI) of treatment and day 21 post-AI, and from control groups on the day of AI and day 21 post-AI for estimation of plasma progesterone, protein, cholesterol, calcium, and inorganic phosphorus profile.Results:The use of CIDR, Ovsynch, and Norgestomet ear implant protocols resulted in 100% estrus induction with conception rates at induced estrus of 60%, 50%, and 50%, and the overall of three cycles as 80%, 80%, and 70%. In untreated anestrus control (n=10), only three cows exhibited spontaneous estrus within 90 days of follow-up and conceived giving the first service and overall conception rates of 66.66% and 30.00%, respectively. In normal cyclic control (n=10), the conception rates at first and overall of three cycles were 50% and 80%. The overall mean plasma progesterone (P4) concentrations in anestrus cows studied on day 0 (initiation), 7 (prostaglandin injection and/or removal of implant), 9 (FTAI) of treatment and on day 21 post-AI revealed that the values on day 7 and 21 were significantly (p<0.01) higher than other two periods in all three groups. The concentrations were significantly (p<0.05) higher in conceived than non-conceived group on day 21 post-AI in CIDR (4.36±0.12 vs. 1.65±0.82 ng/ml) and Ovsynch (4.85±0.62 vs. 1.59±0.34 ng/ml), but not in Norgestomet ear implant (4.50±0.53 vs. 3.02±1.15 ng/ml) or normal cyclic group (5.39±0.67 vs. 3.13±0.37 ng/ml). The cholesterol and protein levels were significantly higher, but not the calcium and phosphorus, in normal cyclic control than in anestrus groups. The influence of treatment days and pregnancy status was not significant for any of the biochemical constituents in any of the groups.Conclusion:Ovsynch and/or CIDR synchronization protocol can be effectively used to improve fertility up to 80% in anestrus cows, as compared to 30% in anestrus control, combined with plasma progesterone to delineate the reproductive status before and after treatment.

Form of supplemental selenium fed to cycling cows affects systemic concentrations of progesterone but not those of estradiol

In areas where soils are deficient in selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Selenium status affects fertility, and the form of Se supplemented to cows affects tissue-specific gene expression profiles. The objective of this study was to determine whether the form of Se consumed by cows would affect follicular growth and the production of steroids. Thirty-three Angus-cross cows that had ad libitum access of a mineral mix containing 35 ppm of Se in free-choice vitamin–mineral mixes as either inorganic (ISe), organic (OSe), or a 50/50 mix of ISe and OSe (MIX) for 180 days were used. After 170 days of supplementation, all cows were injected with 25-mg PGF2α to induce regression of the CL and then monitored for behavioral estrus (Day 0). From Day 4 to Day 8 after estrus, follicular growth was determined by transrectal ultrasonography. On Day 6, cows were injected with PGF2α (20 then 15 mg, 8–12 hours apart) to induce regression of the developing CL and differentiation of the dominant follicle of the first follicular wave into a preovulatory follicle. On Day 8, 36 hours after PGF2α (20 mg), the contents of the preovulatory follicle were aspirated by ultrasound-guided follicular puncture. Blood collected on Days 6 and 8 and follicular fluid collected on Day 8 was analyzed for concentrations of progesterone and estradiol. Form of Se supplemented to cows affected (P = 0.04) the systemic concentration of progesterone on Day 6, but not on Day 8. Form of Se did not affect the systemic concentration of estradiol on Day 6 or Day 8. Form of Se tended to affect (P = 0.07) the concentration of progesterone, but not that of estradiol, in the follicular fluid. Form of Se did not affect diameter of the dominant ovarian follicle on Days 4 to 6, but tended to affect (P = 0.08) the diameter of the preovulatory follicle on Day 8. Our results suggest that form of Se fed to cows affects the production of progesterone but not that of estradiol. Further investigation of organic Se–induced increases in progesterone and potentially the effects of increased progesterone on the establishment of pregnancy, especially in cows of lower fertility, is warranted.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM+ supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Determination of plasma kisspeptin concentrations during reproductive cycle and different phases of pregnancy in crossbred cows using bovine specific enzyme immunoassay

Kisspeptin, a decapeptide and potent secretagogue of GnRH has been emerged recently as a master player in the regulation of reproduction in animals. Determination of kisspeptin in peripheral circulation is, therefore, very important for studying the control of its secretion and its role on reproduction in bovine species, the information on which is not available during any physiological state in this species, may probably be due to non-availability of simple assay procedure to measure the hormone. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for kisspeptin determination in bovine plasma using the biotin–streptavidin amplification system and second antibody coating technique. Biotin was coupled to kisspeptin and used to bridge between streptavidin-peroxidase and the immobilized kisspeptin antiserum in the competitive assay. The EIA was conducted directly in 100μl of unknown bovine plasma. Kisspeptin standards ranging from 0.01 to 25.6ng/100μl/well were prepared in hormone-free plasma. The lowest detection limit was 0.1ng/ml plasma. Plasma volumes for the EIA, viz., 50, 100 and 200μl did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous bovine kisspeptin with kisspeptin standard used. It showed good parallelism with the kisspeptin standard curve. For the biological validation of the assay, plasma kisspeptin was measured in blood samples collected from six non-lactating cyclic cows during entire estrous cycle and from 18 pregnant cows during different stages of pregnancy. The mean plasma kisspeptin concentration during different days of the estrous cycle was different (P<0.001). Three peaks of kisspeptin were recorded, one on a day before appearance of preovulatory LH surge, second at day 6 and third one at day 18 of the estrous cycle. Plasma kisspeptin concentrations increased (P<0.001) from first through last trimester of pregnancy. Kisspeptin concentrations were also measured in different follicular, luteal and placental tissues. Follicular and placental kisspeptin levels increased (P<0.01) during follicular development and with the advancement of pregnancy, respectively. On the other hand, luteal concentrations of kisspeptin decreased (P<0.01) with its developmental process. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma kisspeptin levels in bovine. A wide range of kisspeptin concentrations can be detected during different physiological stages in bovine using this kisspeptin–EIA procedure.

Randomized clinical trial of intrauterine cephapirin infusion in dairy cows for the treatment of purulent vaginal discharge and cytological endometritis

The objectives of this study were to quantify the effect of an intrauterine infusion of cephapirin on reproductive performance at first service of postpartum dairy cows affected by purulent vaginal discharge (PVD) and cytological endometritis (ENDO) using different diagnostic strategies, and to determine if the presence of prolonged anovulation would influence the magnitude of treatment benefit. In total, 2,259 Holstein cows in 28 herds were enrolled in a randomized clinical trial. At 35 (±7) days in milk (DIM), cows were diagnosed with PVD using the Metricheck device (Simcro, Hamilton, New Zealand), with cytological endometritis using endometrial cytology (ENDO-CYTO), and with cytological endometritis using leukocyte esterase (ENDO-LE). Regardless of reproductive tract disease status, cows were randomly assigned to receive an intrauterine cephapirin infusion or to not be treated. Serum progesterone was measured at 35 and 49 (±7) DIM (14 d apart); cows were considered to have prolonged anovulation if progesterone was <1ng/mL at both times. Reproductive events of cows were collected until 200 DIM. Statistical analyses were conducted using multivariable mixed logistic regression models. Intrauterine cephapirin treatment was associated with an increased first-service pregnancy risk in cows diagnosed with PVD (no treatment: 15.4%; treatment: 31.4%), ENDO-CYTO (no treatment: 16.2%, treatment: 24.4%), and ENDO-LE (no treatment: 15.8%; treatment: 25.1%), but not in cows unaffected by any form of reproductive tract disease (no treatment: 34.8%; treatment: 32.6%). Cephapirin treatment was also associated with an increased first-service reproductive performance in cows affected simultaneously by both PVD and ENDO-CYTO (no treatment: 8.7%; treatment: 23.4%). The effect of cephapirin treatment in anovular cows (no treatment: 21.0%; treatment: 26.4%) was numerically lower than in cyclic cows (no treatment: 22.7%; treatment: 34.1%). Overall, an intrauterine infusion of cephapirin improved first-service pregnancy risk in cows with postpartum reproductive tract disease and this effect was influenced by postpartum anovulation status.