Improving pregnancy rates in problem breeder cattle by administration of 15 mg of progesterone on days 3–5 post-mating

Abstract

Days 3–5 post-ovulation is when the embryo is entering the uterus, undergoing genomic activation and progesterone (P₄) increases; therefore, this may be a physiologically important time in the cow. The objective of these experiments was to determine the effect on pregnancy rates through the administration of a low dose of P₄ or altrenogest (ALT; Regumate) on days 3, 4 and 5 of the estrous cycle among repeat breeder (RB) cattle. In Trial 1, RB cows (n=14), that failed to conceive to 3 breedings with a fertile bull, and fertile control cows (n=14) that were an average of 120 days post-partum were used. In Trial 2, RB cows (n=6), RB heifers (n=4) (identified as in Exp 1) and fertile control cows (n=8) that were an average of 135 days post-partum were used. In Exp 1 and 2, all cattle were exposed to a fertile bull for one estrus and the onset of estrus was considered day 0. The respective treatments and blood sample collections were on days 3–5. In Trial 1, RB cows were administered 15 mg of P₄ in 3 ml of ethanol sc and in Trial 2 they were administered 15 mg of ALT (orally) on days 3, 4 and 5. Controls in Trial 1 and 2 were only restrained for blood sample collection to determine plasma P₄ levels using radioimmunoassay (RIA). For both trials, pregnancy rates were empirically lower and similar (P>0.05) between treatments vs. controls. Pregnancy rates for Trial 1 controls were 72% (10/14) and 43% (6/14) for Treatment and for Trial 2 controls were 50% (4/8) and 50% (3/6) for Treatment cows. The mean±SE plasma P₄ levels were higher (P < 0.05) in pregnant controls (1.87±0.2 ng/ml) compared with nonpregnant controls (0.89±0.2 ng/ml) on day 5. In addition, the mean±SE increase in plasma P₄ from day 3–4 was higher (P < 0.05) in pregnant controls (0.42±0.06 ng/ml) compared with nonpregnant controls (0.18±0.11 ng/ml). These resulting pregnancy rates support the hypothesis that P₄ supplementation during this time improve pregnancy rate in RB cattle.

Keywords:

• repeat breeders
• progesterone
• luteal function
• pregnancy

Introduction

Problem breeder (PB) cattle are typically cows that fail to become pregnant in a 60–90 day breeding season or to three or more artificial inseminations (AI). These cows can represent a substantial economic loss to the dairy and beef industry and with increased production becoming a necessity for current cattle production industries new methods to improve pregnancy and calving rates to fewer breeding attempts will be required.

In the past there have been many attempts to improve fertility in high producing cows beginning with an early attempt by ovariectomy and steroid replacement therapy which failed (Hawk et al. 1963). Later researchers began to investigate differences in the physiology of cattle during fertile and non-fertile matings and several were discovered. During infertile matings cows tend to experience prolonged estrus (Erb et al. 1976), delayed pre-ovulatory LH release (Gustafsson et al. 1986; Albihn 1991) and higher cortisol concentrations (Bage et al. 2000; Bage et al. 2001). Furthermore, during an infertile mating cows experience delayed post-ovulatory increases in progesterone (P₄) and corpora lutea (CL) tissue collected from metestrus in PB cows proved less responsive to luteotropin (e.g. LH and PGE₂) stimulation in vitro Shelton et al. 1990). Additionally, the total CL volume in PB cows was lower compared with CL volume collected from fertile cows (Albihn 1991). These findings demonstrate a pattern of delayed luteal function as well as luteal dysfunction in PB cattle.

Additionally, there has been no correlation found between circulating P₄ concentrations and normal embryo morphology in cattle (Linares et al. 1982), however, P₄ concentrations may still be the primary factor involved in the development of a successful pregnancy (Hasler et al. 1980). Using reciprocal embryo transfers among virgin (fertile) heifers and PB cows (transfer embryos from virgin heifers to PB cows and vice versa), it was discovered that PB cows had significantly higher pregnancy rates than virgin heifers (Gustafsson and Larrson 1983). In a subsequent study, embryo survival to day 16–17 was lower in PB cows compared with virgin heifers while, more day 7 embryos collected from virgin heifers survived following transfer to PB cows than PB cow embryos transferred to virgin heifers (Gustafsson 1985). These results demonstrated that the embryos developing from day 1 to day 7 in PB cows had lowered rates of survival than embryos developing from day 1 to day 7 in fertile cattle. A holistic view of these results indicates that the infertility problems associated with PB cows exists within the initial embryo development period (zygote to blastocyst-stage).

These findings indicate deficient uterine secretion or function may be the cause for decreased embryo survival in PB cows. There are known differences in the number of P₄ receptors within the uterus of PB cows (Stanchev et al. 1991), however, there may exist a more likely cause of reduced embryo survival. The hypothesis of this research project was that the timing of the P₄ increase following mating is the limiting factor for improving embryo development in PB cows. Therefore the objective of this study was to determine if an artificially induced rise in P₄, via administration of low doses of P₄ or altrenogest (ALT) on day 3–5 following the onset of estrus (day = 0) would increase pregnancy rates in PB cows.

Materials and methods

Animals

Mature crossbred beef cows (from 5 to 10 years old and from 5 to 8 body condition score) were obtained from the Centre for Reproductive Biology at Louisiana State University, St. Gabriel, Louisiana. All of these cows were treated in accordance with guidelines set forth by the Animal Use Committee for Louisiana State University.

Repeat breeder classification

The breeding programme at the Centre for Reproductive Biology was designed so that a group of mature cows will remain nonpregnant for two years in order to serve on AI and/or embryo transfer (ET) experiments. Should some cows not become pregnant to AI or ET within a two-year period then they are pasture-mated with two fertile bulls for a 65-day breeding period in the spring. The PB cows used in this experiment were those cows that failed to become pregnant to various AI or ET for two years and did not become pregnant during the 65-day breeding season. Since these cows were cycling prior to being placed with the bulls there was an opportunity for three to four matings during each spring breeding season. The PB cows were determined nonpregnant 90 days following the end of the breeding season by ultrasound (Aloka 500-V, Corometrics, Wallingford, CT) examination and were maintained on pasture for an additional three months. Due to the fact that PB cows must be determined in hindsight, the 65-day breeding season was considered the nontreatment period.

The cows classified as fertile were used as control cows for bull fertility and were cows that became pregnant to the same 65-day breeding season one year prior to the respective nontreatment breeding season for the corresponding PB cows. All control cows calved, weaned a live calf and were about 130 days post-partum at the initiation of the experimental (treatment) breeding season.

Estrus detection

Prior to the treatment breeding season, all cows were examined via ultrasonography and only cows free from cystic ovarian disease or reproductive tract abnormalities were used in this experiment. To aid in estrus detection, Heat Watch estrus detection aids (Heat Watch, DDX, Boulder, CO) were used in Trial 1, and Estrus Alert patches (Estrus Alert, Apple Valley, MN) were used in Trial 2. In addition to estrus detection aids, visual observations of 45 minutes twice daily were employed.

Treatment and sample collection

In Trial 1, the treatment breeding season lasted only 25 days to allow for pregnancy rates from one estrus. All cows underwent pregnancy examinations via ultrasonography about 30–33 days following the onset of estrus (day 0 = onset of estrus) and throughout this experiment, a pregnancy was defined as the presence of a fetus with a viable heartbeat. During the treatment breeding season, PB cows were treated with 15 mg of P₄ (Progesterone, Sigma Chemical, St. Louis, MO) dissolved in 3 ml of 100% ethanol (ETOH) (s.c.) on days 3, 4 and 5 after the onset of estrus (estrus = day 0). The control cows received 3 ml of vehicle (ETOH) on the same days.

In Trial 2, the treatment breeding season length was the same as Trial 1. However, in this trial PB cows received 15 mg of ALT orally on days 3, 4 and 5 following the onset of estrus while cows in the control group were brought to the chute and restrained on the same days but received no further treatment.

Radioimmunoassay (RIA) of P₄

In both trials, blood samples were collected immediately prior to treatment via jugular venipuncture, using 10 ml blood tubes containing sodium heparin, on days 3–6 of the estrous cycle. These samples were stored on ice immediately following collection and were later centrifuged at 300× g for 10 minutes. The plasma was collected and stored at 4°C until assay was performed. In Trial 1 and 2 plasma P₄ concentrations were determined using a commercial P₄ assay kit (Diagnostics Systems Laboratory, Webster, TX). The intra- and inter-assay correlation of variation and assay sensitivites were 5%, 10% and 0.05 ng/ml for progesterone. Additionally there was no cross reactivity in this assay with ALT.

Statistical analysis

Pregnancy rates in this experiment were analysed using procedures for categorical data with Chi-square test in SAS (SAS Institute, Inc., Gary, NC). Plasma P₄ concentrations were analysed by repeated measures analysis of variance (ANOVA). Differences in plasma P₄ concentrations among pregnant and nonpregnant cows were analysed using a general linear model (Proc GLM) and Tukey's Test. The rise in P₄ was determined by subtracting the individual cow plasma P₄ value on day 3 from the individual cow plasma P₄ value on day 4. Statistical differences in rise of P₄ were determined using the LSMeans procedure in SAS. Differences with a probability (P) value of 0.05 or less were considered significant in this study.

Results

In both trials there were no differences (P>0.05) in pregnancy rates among control and treatment groups. In Trial 1 the pregnancy rate for the P₄-treated PB cows was 6 of 14 (43%) and for control cows 10 of 14 (72%) and in Trial 2 was 3 of 6 (50%) for ALT-treated PB cows and 4 of 8 (50%) for control cows. However, there was a significant difference (P<0.05) between the pregnancy rate among PB cows during treatment and nontreatment periods with the pooled pregnancy rate for PB cows, regardless of P₄ or ALT treatments being 9 of 20 (45%) compared with 0 of 20 (0%) during the nontreatment period (Table 1).

Table 1. Pooled pregnancy rates for problem breeder cows and control fertile cows during the non-treatment and treatment breeding periods.

Group	Breeding period	n	Treatment	Number pregnant (%)
Problem breeders	Non-Treatment	20	No P4 or altrenogest	0/20 (0%)^a
Problem breeders	Treatment	20	P4 or altrenogest	9/20 (45%)^b
Fertile cows	Treatment	22	Vehicle	14/22 (64%)^b
^a,bValues with differing superscripts within columns represent a significant (P<0.05) difference.

In Trial 1, the mean plasma P₄ concentration was significantly lower (P<0.05) on day 3 in pregnant P₄-treated PB cows (0.25±0.01 ng/ml) compared with non-pregnant P₄-treated PB cows (0.67±0.01 ng/ml) (Figure 1). However, by day 4, the mean plasma P₄ concentration was significantly higher (P<0.05) for pregnant P₄-treated PB cows (2.30±0.7 ng/ml) compared with non-pregnant P₄-treated PB cows (1.22±0.1 ng/ml). This corresponded to a significantly (P<0.05) greater increase (rise) of mean plasma P₄ concentrations from day 3 to day 4 in pregnant P₄-treated PB cows (2.07±0.7 ng/ml) compared with non-pregnant P₄-treated PB cows (0.55±0.12 ng/ml). This pattern was similar to that observed in the control pregnant and nonpregnant cows, however, the greatest rise was seen from day 4–5 (Figure 2).

Figure 1.  Plasma progesterone (P₄) concentrations in pregnant (n=9) and non-pregnant RPB cows (n=11) administered 15 mg of P₄ on days 3, 4 and 5 after the onset of estrus.

Figure 2.  Plasma progesterone (P₄) concentrations in pregnant (n=14) and nonpregnant ‘fertile’ control cows (n=8) on days 3, 4 and 5 after the onset of estrus.

In Trial 2, there were not enough ALT-treated cows to make statistical comparisons so only the mean plasma P₄ concentrations are reported; day 3 (0.68±0.28 ng/ml), day 4 (0.87±0.20 ng/ml), day 5 (2.04±0.44 ng/ml) and day 6 (2.35±0.70 ng/ml). In the control group, pregnant cows had a greater (P<0.05) rise in mean plasma P₄ concentrations from day 3 to day 4 (0.20±0.09 ng/ml) compared with non-pregnant cows (0.44±0.06 ng/ml) Also, the mean plasma P₄ concentrations were greater (P<0.05) in pregnant cows on day 5 (1.87±0.22 ng/ml) compared with nonpregnant cows (0.89±0.16 ng/ml).

Discussion

The results of this experiment demonstrate that administering low doses of P₄ or ALT on days 3–5 post-mating improved pregnancy rates in PB cattle. Although administering progestin to improve pregnancy rates in PB cattle is not a new concept, this study evaluated the importance of the timing of P₄ administration and dosage of P₄. The significant improvement in pregnancy rates among PB cows in this experiment is attributed to the timing and dosage of P₄ or ALT treatment.

A series of physiological events occur on day 3, of the estrous cycle, that are critical to normal embryo development. First, at the 8- to 16-cell stage, the bovine embryo leaves the oviduct to reside within the uterus between day 3 and day 4 (El-Banna and Hafez 1970; Crisman et al. 1980). Also the embryo undergoes genomic activation at this time (Eyestone and First 1986). This is also the same stage of the estrous cycle (day 3) the CL was believed to begin producing significant amounts of P₄ (Henricks et al. 1970; Wettemann and Hafs 1973). It is known that P₄ concentrations can control the rate of embryo transport into the uterus in the mice (Roblero and Garavagno 1979), rat (Forcelledo et al. 1982) and the cow (Crisman et al. 1980) as well as prepare the uterus to support embryo development (Finn and Martin 1967). These findings suggest that day 3 of the estrous cycle may be a time in which a series of critical steps must be completed.

Administration of P₄ anytime from day 1 to day 7 is known to increase uterine secretions (Garrett et al. 1988; Geisert et al. 1992; Barnes 2000) increase the rate of embryo transport from the oviduct into the uterus (Crisman et al. 1980), increase the rate of embryo development as well as embryo length (Garrett et al. 1987), and increase fetal size and crown-rump length later in gestation (Kleeman et al. 1994). Also, when embryos were exposed to advance staged uteri there was an increase in pregnancy rates (Wilmut and Sales 1981). Collectively these studies demonstrate that early increases in P₄ can result in long term beneficial effects for the embryo, however, they involved greater doses of P₄.

In this experiment the natural increase in P₄ occurred from day 4 to day 5 of the estrous cycle in the control cows. This is one day later than was expected, however, pregnant control cows had a significant increase (rise) in plasma P₄ compared with non-pregnant control cows on day 4–5. This greater rise was expected, as this pattern occurred in P₄-treated PB cows from day 3 to day 4. It is difficult to explain why non-pregnant PB cows had significantly lower increases in P₄ from day 3 to day 4 as they were administered the same amount of P₄ as pregnant PB cows. It is possible that in the non-pregnant repeat breeder (RB) cows that the P₄ was metabolised more quickly or bound to carrier proteins rendering it biologically inactive. However, the pattern that emerged was that control and PB cows that experienced significant increases in plasma P₄ from day 3 to day 5 were cows that were pregnant 30 days later.

The reciprocal embryo transfers between virgin heifers and PB cattle studies appear to support these findings. Embryo development was hindered when early development occurred in PB cattle (Gustafsson and Larrson 1983), but occurred normally when early development occurred in virgin-fertile heifers even following transfer on day 7 into PB cattle (Gustafsson 1985). These findings seem to support an early pregnancy model where delayed luteal function could be a possible cause to pregnancy failure. More importantly, these findings show the importance of a healthy, fertile reproductive tract for an early stage embryo.

Previous research has demonstrated that P₄ supplementation initiated prior to day 6 improved pregnancy rates over controls while supplementation after day 6 did not (Mann and Lamming 1999). Unfortunately, the studies involved the administration of a large dose of P₄, up to 100 mg per day in some cases which further complicates the dissection of dosage level effects. Also, the treatment protocols initiated prior to day 5 involved a long-term P₄ supplementation period lasting through the luteal phase. From these studies it can only be concluded that early P₄ is beneficial but not at what time or what level.

In conclusion, supplementation of P₄ or ALT to PB cows on days 3–5 following breeding resulted in increased pregnancy rates. These increased pregnancy rates are likely the result of an induced rise in P₄ between days 3 and 5. These results indicate that an increase in plasma P₄ from day 3 to day 5 of early pregnancy in the cow is beneficial to early embryo development. This effect occurs whether P₄ or ALT is administered. Although the described procedures are off label use of ALT this procedure allows for a practical method of treating PB cows because it is orally administered and possibly substituting with other approved progestins for cattle. These findings may offer a practical and effective method for reducing repeated services among beef and dairy cattle.

Acknowledgements

The authors wish to acknowledge Buzz Yancy of DDX, Inc. (Boulder, CO) for use of HeatWatch® transponders. We also wish would like to acknowledge Gary Sides of Intervet, Inc. (Millsboro, DE) for supplying Regumate used in this research. This research was funded, in part, by Federal Regional Project W-171.