Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have causes other than pregnancy loss. We enrolled Holstein dairy cows ≥50 DIM from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an AI or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a corpus luteum (CL) on d 17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, BCS, and the presence of a CL at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% Bayesian credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92; 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.

Influence of body condition score and its change after parturition on pregnancy rates to fixed-timed artificial insemination in Bos indicus beef cows.

This experiment evaluated pregnancy rates to fixed-time artificial insemination (FTAI) in Bos indicus beef cows according to their body condition score (BCS) at calving and subsequent change until 30 days after FTAI. Non-pregnant, suckling Nelore cows (n = 593 primiparous, 461 secundiparous, and 893 multiparous) were evaluated for BCS at calving and FTAI, and at 30 days after FTAI when cow pregnancy status was verified. Cow BCS at calving was subtracted from BCS recorded at pregnancy diagnosis, and cows classified as those that lost BCS (L), maintained BCS (M), or gained BCS (G) during this period. Cows that calved with BCS ≥ 5.0 had greater (P < 0.01) BCS throughout the experiment, and greater (P < 0.01) pregnancy rates to FTAI compared with cows that calved with BCS < 5.0 (54.8 vs. 34.2%). Pregnancy rates to FTAI were greater (P < 0.01) for G and M cows compared with L cows (50.0%, 47.5%, and 36.0%, respectively), and similar (P = 0.46) between G and M cows. Moreover, pregnancy rates to FTAI in G cows that calved with BCS < 5.0 were less compared with L (tendency; P = 0.08) and M cows (P < 0.01) that calved with BCS ≥ 5.0 (42.2%, 48.3%, and 58.3%, respectively). In summary, pregnancy rates to FTAI were greater in B. indicus cows that calved with a BCS ≥ 5.0 regardless of parity and post-calving BCS change, and greater in M and G cows within those that calved with BCS < 5.0 or ≥ 5.0.

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Increasing the length of an estradiol with progesterone timed artificial insemination protocol with 2 controlled internal drug release devices improves pregnancy per artificial insemination in lactating dairy cows.

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day -11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d -4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d -2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d -12; 25 mg of PG and withdrawal of 1 CIDR on d -4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d -2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13-15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone-based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows.

Size and position of the reproductive tract impacts fertility outcomes and pregnancy losses in lactating dairy cows.

There are multiple factors that contribute to reduced fertility in lactating dairy cows. Recently, a reproductive tract size and position score (SPS) system was developed as a management tool to identify dairy cows with decreased fertility. The aim of this study was to evaluate the association between the SPS on fertility outcomes such as ovulation failure, pregnancy per artificial insemination (P/AI), concentration of pregnancy-associated glycoproteins (PAGs), and pregnancy loss in lactating dairy cows. Primiparous and multiparous lactating Holstein cows (n = 869) were enrolled at two locations. Location 1 (Loc. 1) in Minas Gerais, Brazil (n = 613) and location 2 (Loc. 2) in Agassiz, British Columbia, Canada (n = 256). At the time of AI (d 0), cows were classified as SPS (small [SPS1], medium [SPS2], or large [SPS3] sized reproductive tract) and ovulation failure was determined at 48 h and 7 d post-AI via ultrasonography (Loc. 2 only). Blood samples were collected on d 24 and 31 of gestation for quantification of PAGs and pregnancy diagnosis was performed via ultrasonography at d 31 and 60 post-AI (Loc. 1) and at d 31 ± 3 and 60 ± 3 post-AI (Loc. 2). Cows diagnosed pregnant at d 31 post-AI but not pregnant at d 60 were defined to have undergone late embryonic pregnancy loss. Parity was found to impact SPS (P < 0.01), as primiparous cows had a higher frequency of SPS1 and lower frequency of SPS3 when compared with multiparous cows (SPS1: 42.6 vs. 15.0%; SPS3: 7.0 vs. 22.0%, respectively). Cows classified as SPS3 had greater ovulation failure at 48 h (P = 0.04) and 7 d post-AI (P = 0.05). Cows classified as SPS1 had greater P/AI when compared to SPS2 and SPS3 (45.9 ± 3.3 vs. 37.4 ± 2.6 and 29.1 ± 3.5%, respectively; P = 0.004). There was no interaction between parity and SPS on P/AI. Pregnancy loss between 31 and 60 d post-AI was increased in cows classified as SPS3 compared to SPS2 and SPS1 (24.3 ± 0.05 vs. 11.6 ± 0.02 and 9.4 ± 0.02%, respectively; P = 0.04). Cows classified as SPS1 and SPS2 had greater concentrations of PAGs at 31 d post-AI when compared to SPS3 at both Loc.1 (P < 0.01) and Loc. 2 (P < 0.01). There was no interaction between SPS and pregnancy loss on PAGs at 24 and 31 d post- AI for either Loc. 1 (P = 0.75 and P = 0.76, respectively) or Loc. 2 (P = 0.61 and P = 0.81, respectively). In conclusion, cows that were classified as SPS3 had greater ovulation failure, reduced P/AI, similar concentrations of PAG on d 24, but decreased on d 31, and a greater incidence of pregnancy loss. Thus, size and position of the reproductive tract is associated with fertility and this scoring system could be used to make reproductive management decisions on dairy operations.

Impacts of estrus expression and intensity during a timed-AI protocol on variables associated with fertility and pregnancy success in Bos indicus-influenced beef cows.

This experiment evaluated the impacts of estrus expression and intensity, estimated by physical activity during a timed-AI protocol, on reproductive performance of Bos indicus-influenced beef cows. A total of 290 lactating, primiparous, and multiparous nonpregnant Nelore × Angus cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device (CIDR) on d -11, a 12.5 mg injection of PGF2α on d -4, CIDR removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of eCG on d -2, and timed-AI on d 0. Cows were fitted with a pedometer behind their left shoulder on d -4. An estrus detection patch was attached to the tail-head of each cow on d -2. Pedometer results were recorded on d -2 and 0. Estrus expression was defined as removal of >50% of the rub-off coating from the patch on d 0. Net physical activity during estrus was calculated by subtracting total steps from d -4 to -2 (nonestrus basal activity) from total steps from d -2 to 0 (proestrus + estrus period) of each cow. Cows that did not express estrus were classified as NOESTR. Cows that expressed estrus were ranked by net physical activity; those above the median were classified as HIESTR and the remaining cows as LWESTR. Ovarian ultrasonography was performed on d 0 and 7. Blood was collected on d 0, 7, 20, and 30. Pregnancy status was verified by ultrasonography on d 30. Only data from cows responsive to the estrus synchronization protocol were utilized (NOESTR, n = 59; LWESTR, n = 100; HIESTR, n = 98). Diameter of dominant follicle on d 0, corpus luteum volume on d 7, and plasma P4 concentrations on d 7 were greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR and also greater (P ≤ 0.05) for LWESTR vs. NOESTR. Plasma P4 concentrations on d 0 were greater (P < 0.01) in NOESTR vs. HIESTR and LWESTR and similar (P = 0.93) between HIESTR and LWESTR. Whole blood mRNA expression of myxovirus resistance 2 on d 20 was greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR, and similar (P = 0.72) between LWESTR and NOESTR. Pregnancy rates were less (P ≤ 0.04) in NOESTR vs. HIESTR and LWESTR (52.4%, 68.9%, and 73.5%, SEM = 7.2), and similar (P = 0.57) between HIESTR and LWESTR. Hence, expression of estrus during a timed-AI protocol improved ovarian dynamics and pregnancy success, whereas estrus intensity modulated key biological markers associated with fertility but not pregnancy rates in B. indicus-influenced cows beef cows.

Effect of preovulatory follicle maturity on pregnancy establishment in cattle: the role of oocyte competence and the maternal environment

Reproductive technologies to synchronize estrus and ovulation in cattle have enhanced the ability to practically utilize artificial insemination to increase both genetic merit and reproductive management of beef and dairy herds. The ability to successfully synchronize a follicular wave and ovulation, in heifers and cows, has improved substantially in recent years. Consequently, pregnancy rates to a single fixed-time artificial insemination (FTAI) can approximate that of insemination following spontaneous estrus. Despite these advances, a subset of heifers and cows often has a physiologically immature dominant follicle at the time of GnRH-induced ovulation. These animals will exhibit reduced pregnancy rates and decreased embryonic survival if a pregnancy happens to become established. The physiological mechanisms underlying the preceding decreased fertility have been a focus of our laboratories and may include an effect of the follicular microenvironment on both oocyte competence and the maternal environment. Oocytes must have adequate opportunity to complete cytoplasmic and molecular maturation during the final stages of oocyte maturation that occur within the preovulatory follicle. Follicular status, during the proestrus period, must be such that adequate circulating concentrations of estradiol are present before FTAI to increase oviductal transport of gametes and enhance both the luteinizing capacity of granulosa cells and progesterone receptor population in the post-ovulatory uterus. Following ovulation, the follicles transformation to a functional corpus luteum to secrete adequate amounts of progesterone is essential for the establishment of pregnancy. The physiological status of the preovulatory follicle, prior to FTAI, greatly affects the concepts discussed above and has an important impact on pregnancy establishment and maintenance in cattle.(AU)

Markers of pregnancy: how early can we detect pregnancies in cattle usingpregnancy-associated glycoproteins (PAGs) and microRNAs?

Pregnancy detection has evolved over the last few decades and the importance of early pregnancy detection is critical to minimize the amount of time a cow spends not pregnant. Embryonic mortality (EM) is generally considered to be the primary factor limiting pregnancy rates in cattle and occurs early (

Effect of preovulatory follicle maturity on pregnancy establishment in cattle: the role of oocyte competence and the maternal environment

Reproductive technologies to synchronize estrus and ovulation in cattle have enhanced the ability to practically utilize artificial insemination to increase both genetic merit and reproductive management of beef and dairy herds. The ability to successfully synchronize a follicular wave and ovulation, in heifers and cows, has improved substantially in recent years. Consequently, pregnancy rates to a single fixed-time artificial insemination (FTAI) can approximate that of insemination following spontaneous estrus. Despite these advances, a subset of heifers and cows often has a physiologically immature dominant follicle at the time of GnRH-induced ovulation. These animals will exhibit reduced pregnancy rates and decreased embryonic survival if a pregnancy happens to become established. The physiological mechanisms underlying the preceding decreased fertility have been a focus of our laboratories and may include an effect of the follicular microenvironment on both oocyte competence and the maternal environment. Oocytes must have adequate opportunity to complete cytoplasmic and molecular maturation during the final stages of oocyte maturation that occur within the preovulatory follicle. Follicular status, during the proestrus period, must be such that adequate circulating concentrations of estradiol are present before FTAI to increase oviductal transport of gametes and enhance both the luteinizing capacity of granulosa cells and progesterone receptor population in the post-ovulatory uterus. Following ovulation, the follicle’s transformation to a functional corpus luteum to secrete adequate amounts of progesterone is essential for the establishment of pregnancy. The physiological status of the preovulatory follicle, prior to FTAI, greatly affects the concepts discussed above and has an important impact on pregnancy establishment and maintenance in cattle.

Effect of progesterone concentrations, follicle diameter, timing of artificial insemination, and ovulatory stimulus on pregnancy rate to synchronized artificial insemination in postpubertal Nellore heifers.

Two experiments were designed to evaluate the effects of treatments with low versus high serum progesterone (P4) concentrations on factors associated with pregnancy success in postpubertal Nellore heifers submitted to either conventional or fixed timed artificial insemination (FTAI). Heifers were synchronized with a new controlled internal drug release device (CIDR; 1.9 g of P4 [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus 2 mg of estradiol (E2) benzoate on Day 0 and 12.5 mg of prostaglandin F2α on Day 7. In experiment 1 (n = 723), CIDR were removed on Day 7 or 9 and heifers were inseminated after estrus detection. In experiment 2 (n = 1083), CIDR were all removed on Day 9 and FTAI was performed either 48 hours later in heifers that received E2 cypionate (ECP) on Day 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH (100 µg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P4 concentrations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment 1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day 7, 3.76 ± 0.08 days vs. Day 9, 2.90 ± 0.07; P < 0.01) and improved conception (Day 7, 57.1% vs. Day 9, 65.8%; P = 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P = 0.04). In experiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus (E48, 40.9%(a); G54, 17.1%(c); and G72, 32.0%(b)), but the pregnancy rate was not affected (P = 0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3 increased follicle diameter at FTAI (CIDR1, 11.07 ± 0.10 vs. CIDR3, 11.61 ± 0.10 mm; P < 0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception (CIDR1, 42.2 vs. CIDR3, 45.1%; P = 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P = 0.11). In conclusion, length of treatment with P4 affected the fertility of heifers bred based on estrus detection. When the heifers were submitted to FTAI protocol, follicle diameter at FTAI (≤10.7 mm, 23.6%; 10.8-15.7 mm, 51.5%; ≥15.8 mm, 30.0%; P < 0.01) was the main factor that affected conception and pregnancy rates.