The Induction of a Secondary Corpus Luteum on Day 12 Post‐Ovulation can Delay the Time of Luteolysis in High‐Producing Holstein Cows

Luteolysis before the time of maternal recognition of pregnancy is one cause of low fertility in high‐producing dairy cows. The objective of this study was to assess whether induction of a secondary corpus luteum (CL) late in the luteal phase would delay the time of luteolysis. Twenty high‐producing Holstein cows were synchronized to ovulation (Day 0) with the Ovsynch protocol and received hCG (1500 IU im) on Day 12. Corpora lutea formation (as evaluated by ultrasonography) and plasma P4 concentrations were monitored from Days 4 to 36. hCG treatment induced the formation of one secondary CL (CL2) in 11 of 20 cows (55%) from the dominant follicle (mean diameter: 14.2 ± 0.9 mm) of two‐wave (3/11) and three‐wave (8/11) cycles. The maximal diameter of the CL2 (23.3 ± 1.9 mm) was reached approximately 6 days after hCG treatment and was correlated with its structural lifespan (p < 0.01). Cows that formed a CL2 after hCG had higher mean plasma P4 concentrations on Day 14 (+4.5 ng/ml) and Day 18 (+3.0 ng/ml) compared with cows without CL2 (p < 0.05). The structural regression of CL2 begun approximately 8 days after that of the CL1, and the median time at which the first drop in circulating P4 levels occurred was later in cows that formed a CL2 than in those that did not (Day 26 vs Day 18; p < 0.01). Thus, the induction of a CL2 by hCG on Day 12 might reduce the risk of premature luteolysis in high‐producing dairy cows after insemination.     

Factors Affecting Synchronization and Conception Rate after the Ovsynch Protocol in Lactating Holstein Cows

Objectives were to evaluate risk factors affecting ovulatory responses and conception rate to the Ovsynch protocol. Holstein cows, 466, were submitted to the Ovsynch protocol [day 0, GnRH‐1; day 7, prostaglandin (PG) F_2α; day 9, GnRH‐2] and 103 cows were inseminated 12 h after GnRH‐2. Information on parity, days in milk at GnRH‐1, body condition, milk yield, exposure to heat stress, pre‐synchronization with PGF_2α and the use of progesterone insert from GnRH‐1 to PGF_2α was collected. Ovaries were scanned to determine responses to treatments. Overall, 54.7%, 10.6%, 2.2%, 81.1%, 9.0%, 91.5% and 36.9% of the cows ovulated to GnRH‐1, multiple ovulated to GnRH‐1, ovulated before GnRH‐2, ovulated to GnRH‐2, multiple ovulated to GnRH‐2, experienced corpus luteum (CL) regression and conceived, respectively. Ovulation to GnRH‐1 was greater in cows without a CL at GnRH‐1, cows with follicles >19 mm and cows not pre‐synchronized with PGF_2α 14 days before GnRH‐1. Multiple ovulations to GnRH‐1 increased in cows without CL at GnRH‐1 and cows with follicles ≤19 mm at GnRH‐1. Ovulation before GnRH‐2 was greater in cows without CL at PGF_2α. Ovulation to GnRH‐2 increased in cows that received a progesterone insert, cows with a CL at GnRH‐1, cows with follicles not regressing from the PGF_2α to GnRH‐2, cows with larger follicles at GnRH‐2, cows that ovulated to GnRH‐1 and cows not pre‐synchronized. Multiple ovulations after GnRH‐2 increased in cows with no CL at GnRH‐1, multiparous cows and cows that multiple ovulated to GnRH‐1. Conception rate at 42 days after AI increased in cows with body condition score > 2.75 and cows that ovulated to GnRH‐2. Strategies that optimize ovulation to GnRH‐2, such as increased ovulation to GnRH‐1, should improve response to the Ovsynch protocol.     

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds

When European Union regulations restricted the use of estrogenic compounds in food‐producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F_2α (PGF_2α) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin‐releasing hormone of Ovsych and the last PGF_2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca²⁺ and total protein in cervical mucus in all three hormone‐treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Nitric oxide in bovine corpus luteum: Possible mechanisms of action in luteolysis

Although prostaglandin (PG) F_2α is considered as the principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intraovarian factors. Among them, nitric oxide (NO) seems to play a mandatory role in luteolysis. In this article we review the background and current status of work on possible roles of NO in the CL function, based on available information and our own experimental data. NO is produced in all three main types of bovine CL cells: steroidogenic, endothelial and immune cells. PGF_2α and some luteolytic cytokines (tumor necrosis factor, interferon) increase NO production and stimulate NO synthase expression in the bovine CL. NO inhibits progesterone production, stimulates the secretion of PGF_2α and leukotriene C4, reduces the number of viable luteal cells and, finally, participates in functional luteolysis. NO induces the apoptotic death of CL cells by the modulation of bcl‐2 family gene expression and the stimulation of caspase‐3 gene expression and activity. However, this simple molecule shows both luteolytic and luteotropic actions during the estrous cycle in ruminants. The aim of this overview is to present and discuss the recent findings crucial for understanding NO role in the process of CL regression in cattle.     

Local mechanisms for luteolysis in the cow: Novel roles of vasoactive substances in the luteolytic cascade within the corpus luteum

The corpus luteum (CL) in the estrous cycle in the cow is a dynamic organ which has a lifespan of approximately 17–18 days. As the CL matures, the steroidogenic cells establish contact with many capillary vessels and the CL is composed of a large number of vascular endothelial cells that can account for up to 50% of the bovine CL. Furthermore, luteal cells and endothelial cells secrete several vasoactive substances such as prostaglandin F_2α (PGF_2α), endothelin‐1 and angiotensin II. These vasoactive substances also function in regulating progesterone secretion in an autocrine/paracrine manner in the CL. The blood vessels and endothelial cells in the CL therefore have an essential role in the luteal function in the cow. Endometrial PGF_2α, the primary luteolysin in the cow, stimulates luteal vasoactive substances during luteolysis. Moreover, luteal vasoactive substances may have key roles in the regulation of luteolysis to induce vasodilatation, vasoconstriction and angiolysis. This review describes the current concept for possible roles of vasoactive substances in the luteolytic cascade within the bovine CL.     

Pregnancy Rates in Angus Cross Beef Cows Bred at Observed Oestrus With or Without Second GnRH Administration in Fixed‐Time Progesterone‐Supplemented Ovsynch and CO‐Synch Protocols

Crossbred cows (n = 1073) from five locations had oestrous cycles synchronized with 100 μg of GnRH IM and insertion of controlled internal drug release device (CIDR) on Day 0 followed by 25 mg of PGF_2α IM and CIDR removal on Day 7. Kamar^® patches were placed on all cows at CIDR removal. Cows were observed three times daily for oestrus after PGF_2α administration. In the Ovsynch‐CIDR group, cows detected in oestrus (n = 193) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 80 received and 113 did not receive a second GnRH at 48 h after PGF_2α. Cows (n = 345) not detected in oestrus received a second GnRH at 48 h after PGF_2α on Day 9, and fixed‐time AI 16 h after the GnRH on Day 10. In the CO‐Synch‐CIDR group, cows detected in oestrus (n = 224) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 79 received and 145 did not receive a second GnRH at 64 h after PGF_2α. Cows (n = 311) not detected in oestrus received a second GnRH on Day 10 at the time of AI, 64 h after PGF_2α. The AI pregnancy rates were not different between the Ovsynch‐CIDR and CO‐Synch‐CIDR groups (p = 0.48). There were no differences in the AI pregnancy rates for cows inseminated at a fixed time (p = 0.26) or at detected oestrus (p = 0.79) between the treatment groups. Among cows inseminated in oestrus, there were no differences in the AI pregnancy rates between cows that received or did not receive the second GnRH (p = 0.47). In conclusion, acceptable AI pregnancy rates can be achieved with or without inclusion of oestrus detection in the Ovsynch‐CIDR and CO‐Synch‐CIDR protocols. Among cows detected in oestrus, cows that received a second GnRH yielded similar pregnancy rates when compared with cows that did not receive the second GnRH.     

Effect of exogenous progesterone administration on luteal sensitivity to PGF during the early development of the corpus luteum in mares and cows

The objective of this study was to determine the effect of exogenous progesterone administration at ovulation and during the early development of the CL, on its future sensitivity to a single administration of PGF2a in mares and cows. Horse Retrospective reproductive data from an equine clinic in the UK during three breeding seasons were used. Mares were divided into: control group, cycles with single ovulations; double ovulation group cycles with asynchronous double ovulations; and PRID group: cycles with single ovulations and treatment with intravaginal progesterone device (CIDR) immediately after the ovulation. All mares were treated with d‐cloprostenol (PGF) at either: (i) 88 hr; (ii) 96 hr; (iii) 104 hr; or (iv) 112 hr after the last ovulation. Cattle A total of nine non‐lactating Holstein cows were used. All cows were administered PGF14 d apart and allocated to one of two groups control group GnRH was administered 56 hr after the second PGF administration. CIDR group CIDR was inserted at the same time of GnRH administration. All cows were administered PGF at 120 hr post‐ovulation. The complete luteolysis rate of mares with double ovulation (66.7%) and those treated with exogenous progesterone (68.4%) was significantly higher than the rate of mares with single ovulation (35.6%) at 104 hr. In the cow, however, the treatment with CIDR did not increase the luteolytic response in cows treated at 120 hr post‐ovulation. In conclusion, the degree of complete luteolysis can be influenced by increasing the concentration of progesterone during the early luteal development in mares.     

Presence of multiple corpora lutea affects the luteolytic response to prostaglandin F2α in lactating dairy cows

Since the 1970s, luteolytic doses used for synchronizing estrus in dairy cattle have remained unchanged. This study aimed to evaluate the dose-response effect of prostaglandin F_2α (PGF_2α), which is used for synchronizing estrus, and subsequent fertility in cows with two or more corpora lutea (CL). The study population consisted of 1,683 cows with a single CL (1CL), 501 cows with multiple CL receiving a single dose of PGF_2α (2CL1), and 252 cows with multiple CL receiving a 1.5 × PGF_2α dose (2CL1.5). Cows with a single CL (n = 1,245) showed estrus significantly (P < 0.01) earlier (3.01 ± 1.23 days; mean ± SD) than cows with multiple CL (n = 287; 3.33 ± 1.69 days). Using 1CL cows as reference, the odds ratio (OR) for the estrus response in 2CL1 cows was 0.13 (P < 0.0001), whereas the ORs for estrus response and pregnancy of 2CL1.5 cows were 1.8 (P = 0.0001) and 1.7 (P = 0.001), respectively. Based on the results for only the 2CL1 cows, the OR for the estrus response was 0.7 (P = 0.01) for cows producing ≥ 45 kg of milk at treatment, compared to the remaining cows producing < 45 kg of milk. Our results showed that the presence of multiple CL reduced the estrus response to that induced by a single PGF_2α dose and milk production was inversely associated with this response, whereas an increased PGF_2α dose improved the estrus response. Therefore, an increase in the standard PGF_2α dose is recommended.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells,Smooth Muscle Cells and Granulosa Cells

Prostaglandin F_2α (PGF_2α) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF_2α receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF_2α and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage‐dependent and the site‐restricted effect of PGF_2α and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co‐cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully‐luteinized GC. PGF_2α stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix‐cultures of EC and SMC with fully‐luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF_2α at 1 h only when EC was cultured together with fully‐luteinized GC but not with luteinizing GC. In all co‐cultures, PGF_2α did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully‐luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF_2α.     

Hepatic steroid inactivating enzymes,hepatic portal blood flow and corpus luteum blood perfusion in cattle

Production from the corpus luteum (CL) and/or hepatic steroid inactivation impacts peripheral concentrations of P4, which can alter reproductive performance. Our primary objective was to examine hepatic steroid inactivating enzymes, portal blood flow, and luteal blood perfusion at 10 days post‐insemination in pregnant versus non‐pregnant beef and dairy cows. Twenty early lactation Holstein cows and 20 lactating commercial beef cows were utilized for this study. At day 10 post‐insemination, hepatic portal blood flow and CL blood perfusion were measured via Doppler ultrasonography. Liver biopsies were collected and frozen for later determination of cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), uridine diphosphate‐glucuronosyltransferase (UGT) and aldo‐keto reductase 1C (AKR1C) activities. Pregnancy was determined at day 30 post‐insemination and treatment groups were retrospectively assigned as pregnant or non‐pregnant. Data were analyzed using the mixed procedure of SAS. Steroid metabolizing enzyme activity was not different (p > .10) between pregnant versus non‐pregnant beef or dairy cows. Hepatic portal blood flow tended (p < .10) to be increased in pregnant versus non‐pregnant dairy cows. Luteal blood perfusion was increased (p < .05) in pregnant versus non‐pregnant dairy cows. Pregnant dairy cows appear to have an increased rate of hepatic clearance of P4 in combination with increased synthesis from the CL. This could account for the lack of difference in peripheral P4 concentrations between pregnant and non‐pregnant dairy cows. This study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.     

Effect of Initial GnRH and Duration of Progesterone Insert Treatment on the Fertility of Lactating Dairy Cows

The study compared response to prostaglandin F2α (PG), synchrony of ovulation and pregnancy per AI (P/AI) in a 5‐ vs a 7‐day Ovsynch + PRID protocol and investigated whether the initial GnRH affects P/AI in lactating dairy cows. Two hundred and seventy‐six cows (500 inseminations) were assigned to one of four timed‐AI (TAI) protocols: (i) PRID‐7G; 100 μg GnRH im, and a progesterone‐releasing intravaginal device (PRID) for 7 days. At PRID removal, PG (500 μg of cloprostenol) was given im. Cows received the second GnRH treatment at 60 h after PRID removal and TAI 12 h later. (ii) PRID‐5G; as PRID‐7G except the duration of PRID, treatment was 5 days and PG was given twice (12 h apart). (iii) PRID‐7NoG; as PRID‐7G except the initial GnRH, treatment was omitted. (iv) PRID‐5NoG; as PRID‐7NoG except the duration of PRID, treatment was 5 days. Response to treatments and pregnancy status at 32 and 60 days after TAI was determined by ultrasonography. The percentage of cows ovulating before TAI was greatest in PRID‐7G (17.1%), and the percentage of cows that did not have luteal regression was greatest in PRID‐5G (9.5%). The overall P/AI at 32 and 60 days did not differ among TAI protocols. However, during resynchronization, cows subjected to the 5‐day protocols had greater (p < 0.05) P/AI (45.3% vs 33.6%) than cows subjected to the 7‐day protocols. Pregnancy loss between 32 and 60 days tended (p = 0.10) to be greater in cows that did not receive initial GnRH (14.8%) compared to those that received GnRH (8.2%). In conclusion, the PRID‐5G protocol resulted in fewer cows responding to PG, but P/AI did not differ among TAI protocols. A 5‐day protocol resulted in more P/AI in resynchronized cows, and cows that did not receive initial GnRH tended to experience more pregnancy losses.     

Gonadotropin‐Releasing Hormone Administration to Dairy Cows without a Corpus Luteum 4 Weeks after Calving Increases Reproductive Performance

This field study investigated whether the administration of a single dose of gonadotropin‐releasing hormone (GnRH) to dairy cows without a corpus luteum (CL) 4 weeks after calving can improve reproductive performance. Holstein dairy cows underwent ultrasonography to assess the presence of ovarian structures at 29.2 ± 5.2 days post‐partum, and cows were divided into two main groups based on the presence (CL group, n = 230) or absence (non‐CL group, n = 460) of a CL. The non‐CL group was further randomly divided into two subgroups based on the administration of GnRH (non‐CL GnRH group, n = 230) or no GnRH (non‐CL control group, n = 230). Subsets of cows from non‐CL control (n = 166) and non‐CL GnRH (n = 175) groups received a second ultrasonography at 44.5 ± 5.4 days post‐partum to assess CL formation. The percentage of cows with CL at the second ultrasonography was greater in the non‐CL GnRH group (70.9%) than in the non‐CL control group (53.0%, p = 0.0006). The hazard of the first post‐partum insemination by 150 days in milk (DIM) was higher in the CL group than in the non‐CL control group (hazard ratio [HR]: 1.36, p = 0.001). The probability of a pregnancy to the first insemination was higher in non‐CL GnRH (odds ratio [OR]: 1.50, p = 0.04) and CL groups (OR: 1.55, p = 0.03) compared to the non‐CL control group. Furthermore, the hazard of pregnancy by 210 DIM was higher in non‐CL GnRH (HR: 1.30, p = 0.01) and CL (HR: 1.51, p = 0.0001) groups than in the non‐CL control group. In conclusion, administration of GnRH to dairy cows without a CL 4 weeks after calving was associated with an increase in ovulation and improved reproductive performance.     

Ovarian response to prostaglandin F2α in lactating dairy cows: A clinical update

Prostaglandin F_2α (PGF_2α) and its analogs are used to induce luteolysis in estrus synchronization programs to terminate unwanted pregnancies or to promote ovulation in certain cow subpopulations. In the past few decades, the luteolytic dose of PGF_2α has remained unchanged. This review explores the clinical implications of increasing the standard dose for these applications in high-producing dairy cows. Ultrasonography may assist in selecting the most appropriate PGF_2α dose and improve the results. A reference has been used for PGF_2α for promoting ovulation in herds showing poor reproductive performance.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

Corpus Luteum Development and Function after Supplementation of Long‐Acting Progesterone During the Early Luteal Phase in Beef Cattle

Strategic supplementation of P4 may be used to increase conception rates in cattle, but timing of supplementation in relation to ovulation, mass of supplementary P4 and formulation of the P4‐containing supplement has not been determined for beef cattle. Effects of supplementation of long‐acting progesterone (P4) on Days 2 or 3 post‐ovulation on development, function and regression of corpus luteum (CL) were studied in beef cattle. Cows were synchronized with an oestradiol/P4‐based protocol and treated with 150 or 300 mg of long‐acting P4 on Day 2 or 3 post‐ovulation (6–7 cows/group). Colour‐doppler ultrasound scanning and blood sample collection were performed from Day 2–21.5. Plasma P4 concentrations were greater (p < 0.05) from Day 2.5–5.5 in the Day 2‐treated groups and from Day 3.5–5.5 in the Day 3‐treated cows than in the control group. CL area and blood flow during Day 2–8.5 did not differ (p > 0.05) among groups, suggesting no effect of P4 treatment on luteal development. The frequency of cows that began luteolysis before Day 15 was greater (p < 0.04) in cows treated with 300 mg than in the controls, but there were no differences between non‐treated and 150 mg‐treated cows. The interval from pre‐treatment ovulation to functional and structural luteolysis was shorter (p < 0.01) in the combined P4‐treated groups than in the control cows. In conclusion, was showed for the first time that long‐acting P4 supplementation on Day 2 or 3 post‐ovulation increases P4 concentrations for ≥3 day, has no effect on luteal development, but anticipates the beginning of luteolysis in beef cattle.     

IFN‐τ Acts in a Dose‐Dependent Manner on Prostaglandin Production by Buffalo Endometrial Stromal Cells Cultured in vitro

Interferon‐τ (IFN‐τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂). PGF_2α is responsible for the luteolysis; however, PGE₂ favours establishment of pregnancy by its luteoprotective action. In this study, the dose‐response effect of recombinant bovine IFN‐τ (rbIFN‐τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN‐τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN‐τ treatment enhanced the secretion of both PGE₂ and PGF_2α, and also its ratio in a strict dose‐dependent manner with a significant increase (p < 0.01) in PGE₂ production at 1 μg/ml dose of rbIFN‐τ and maximal stimulation for both PG was observed at 10 μg/ml. Further, both PG production and its ratio were increased significantly (p < 0.01) in a time‐dependent fashion in all the groups at 6, 12 and 24 h post‐treatment with highest level achieved at 24 h as compared with control. Absolute levels of PGE₂ remained higher than PGF_2α indicating PGE₂ as the major PG produced by endometrial stromal cells. The dose‐dependent response of rbIFN‐τ signifies the importance of optimum concentration of IFN‐τ for the embryonic development especially during the critical period to establish successful pregnancy.     

Concentrations of retinol and tocopherols in the milk of cows supplemented with conjugated linoleic acid

This study was performed to investigate the hypothesis that supplementation of conjugated linoleic acid (CLA) changes the concentrations of retinol and tocopherols in the milk of cows. To investigate this hypothesis, Holstein cows received daily from 3 weeks ante‐partum to 14 weeks post‐partum either 172 g of a CLA‐free rumen‐protected control fat (control group, n = 20) or the same amount of a rumen‐protected CLA fat, supplying 4.3 g of cis‐9, trans‐11 CLA and 3.8 g of trans‐10, cis‐12 CLA per d (CLA group, n = 20). Milk samples (collected at weeks 1, 3, 5, 8 and 11 of lactation) were analysed for retinol, α‐ and γ‐tocopherol concentrations. Milk of cows supplemented with CLA had higher concentrations of retinol (+34%), α‐tocopherol (+44%) and γ‐tocopherol (+21%) than milk of control cows (p < 0.05). The daily output of these vitamins via milk was also greater in cows of the CLA group than in cows of the control group (+36, 50 and 24% for retinol, α‐tocopherol and γ‐tocopherol, respectively, p < 0.05). In agreement with higher concentrations of tocopherols, concentrations of thiobarbituric acid‐reactive substances, determined in milk of week 5, were lower in cows of the CLA group than in control cows, indicative of a lower susceptibility of milk lipids to peroxidation. Plasma concentrations of retinol and α‐tocopherol, determined at 1 and 5 weeks post‐partum, were not different between the two groups of cows. In conclusion, this study shows that supplementing dairy cows with a moderate amount of CLA causes an increase of the concentrations of vitamins A and E in the milk and results in an increased output of those vitamins via milk. These effects might be beneficial with respect to the nutritional value of dairy products and the susceptibility of milk fat to oxidative deterioration.     

Fertility in Dairy Cows After Artificial Insemination Using Sex‐Sorted Sperm or Conventional Semen

The aim of this study was to compare pregnancy per artificial insemination (P/AI) after timed AI with sex‐sorted sperm (SS) or conventional semen (CS) in lactating dairy cows. Cyclic cows (n = 302) were synchronized by Ovsynch and randomly assigned into two groups at the time of AI. Cows with a follicle size between 12 and 18 mm and clear vaginal discharge at the time of AI were inseminated with either frozen‐thawed SS (n = 148) or CS (n = 154) of the same bull. A shallow uterine insemination was performed into the uterine horn ipsilateral to the side of probable impending ovulation. Pregnancy per AI on Day 31 tended (p = 0.09) to be less for SS (31.8%) than CS (40.9%). Similarly, P/AI on Day 62 was less (p = 0.01) for cows inseminated with SS (25.7%) compared with CS (39.0%). The increased difference in fertility between treatments from Days 31 to 62 was caused by the greater (p = 0.02) pregnancy loss for cows receiving SS (19.2%) than CS (4.8%). Cow parity (p = 0.02) and season (p < 0.01) when AI was performed were additional factors affecting fertility. Primiparous cows had greater P/AI than multiparous cows both on Day 31 (41.7% vs 25.0% in SS and 53.0% vs 31.8% in CS groups) and on Day 62 (33.3% vs 20.5% in SS and 48.5% vs 31.8% in CS groups). During the hot season of the year, P/AI on Day 31 was reduced (p = 0.01) in the SS group (19.6%) when compared with the rates during the cool season (38.1%). In conclusion, sex‐sorted sperm produced lower fertility results compared to conventional semen even after using some selection criteria to select most fertile cows.