Introduction

Low fertility in dairy cows is due to high incidence of early embryo death (^{Diskin et al., 2006}). One of the causes is related to delay in embryo development and the subsequent

reduction in interferon- τ secretion, causing failure of luteolysis inhibition (^{Mann and Lamming, 2001}). Delay in embryo development and the subsequent reduction in interferon-τ secretion, causing failure of luteolysis inhibition (Mann and Lamming, 2001). Delay in embryo development may be determined by diminishing progesterone serum concentrations (Mann and Lamming, 2001; ^{Leroy et al, 2008}). High-producing dairy cows have lower progesterone levels because the corpus luteum produces less progesterone (^{Stronge et al., 2005}) and because they catabolize steroid hormones more rapidly (^{Sangsritavong et al., 2002}; ^{Vasconcelos et al, 2003}).

Progesterone addition has favored embryo development and secretion of interferon- τ (^{Mann and Lamming, 1999}; ^{Mann et al, 2006}; ^{Carter et al., 2008}). In practice, there are several approaches to increasing progesterone serum levels and, thus, improving the proportion of pregnant cows (conception rate). Progesterone is administered using implants or intravaginal devices with variable results (Mann and Lamming, 1999; ^{Larson et al, 2007}; ^{Friedman et al., 2012}). Increased progesterone blood concentrations and conception rate are achieved by inducing formation of an accessory corpus luteum by injecting hCG five days after insemination (^{Santos et al, 2001}; ^{Urzúa et al, 2009}; ^{Nascimento et al, 2013}).

Response to progesterone depends on the time elapsed after insemination. Thus, if progesterone is added between days 5 and 9 after insemination, there is a favorable effect on embryo development and interferon- τ secretion. In contrast, the same treatment has no effect when applied between days 12 and 16 (^{Mann and Lamming, 1999}; Mann et al., 2006). Moreover, there is a positive association between concentrations of progesterone during days 5 to 7 of the estrous cycle and embryo survival (^{Stronge et al, 2005}). ^{Macmillan and Peterson (1993)} observed that insertion of a progesterone release device from day 6 to 8 post-insemination increased fertility, but after this time, insertion had no favorable effect. These observations suggest that there is a physiological period in which an increase in progesterone serum levels can favor embryo survival and, consequently, fertility. One way to increase progesterone blood concentration is through the use of intravaginal devices that release this hormone. These devices, however, result in marginal blood progesterone increases of 0.8 to 1 ng mL^-1 (^{Cerri et al., 2009}; ^{Lima et al., 2009}) in high-producing cows. An injection of 500 mg progesterone in lactating cows increases blood concentrations of this hormone more than 1 ng mL^-1 and lasts 48 to 72 h (^{Flores et al, 2013}). The objective of this study was to test whether an intramuscular injection of 500 mg progesterone 5 d after insemination increases conception rate in dairy cows.

Materials and Methods

The study was conducted from August to December in a dairy herd located on the central high plateau of Mexico. The climate of the region is temperate subhumid with an annual average temperature of 15.5 °С and mean annual rainfall of 861 mm.

Results and Discussion

The conception rate was no afected by injection of progesterone (Table 1). Injection 5 d after insemination caused a transient increase in blood during the following 48 h, relative to the control cows (Figure 1; p≤0.05). The interactions between variables were not significant (p>0.10). In this study we proposed that a temporary increase in the progesterone serum levels during early diestrus could favor fertility. This was supported by studies that found a positive association between progesterone concentrations from days 5 to 9 and embryo development (^{Macmillan and Peterson, 1993}; ^{Stronge et al, 2005}; ^{Mann et al, 2006}). The absence of effect of the proposed treatment in this study may be due to the method of progesterone administration. In the cited studies (Macmillan and Peterson, 1993; Mann et al, 2006), progesterone was administered using an intravaginal device, which caused a sustained increase in blood concentrations. In our study, the increase observed was abrupt during the 24 h following the injection and during the subsequent 48 h there was a rapid fall. To maintain high levels of progesterone during the period referred to in the cited studies, a second injection 48 h later or a different mode of application, subcutaneous for example, would permit slower absorption of the hormone.

Table 1 Odds ratio for conception probability, according to the variables in the logistic regression model. 

*Conception rate.

**Only first service cows are included.

Figure 1 Blood progesterone concentrations (mean ± standard error) of cows treated with 500 mg intramuscular progesterone (-О-) at day five after insemination and of control cows (-□-). 

The treatments based on increasing blood progesterone concentrations, by supplementation or by inducing formation of an accessory corpus luteum, have had varying results, associated with characteristics of sub-populations of treated cows. ^{Friedman et al. (2012)} observed that progesterone supplementation increased fertility only in cows with a low body condition score and in cows with postpartum reproductive pathologies. ^{Santos et al. (2001}) and ^{Urzúa et al. (2009}) observed that cows that responded favorably to treatment with hCG 5 d after insemination were first service cows and, particularly, those that lost body condition in the 30 d following insemination. In our study, the effect of the interaction treatment X body condition was not observed; body condition dynamics after treatment was not quantified (Table 1).

Days in milk, number of parturitions, type of estrus and characteristics of Puerperium affected conception rate, but the interaction of these variables with the treatment (p≥0.10) was not observed. Likelihood of pregnancy was lower in cows inseminated 90 to 150 and > 150 d in milk than in cows inseminated the first 90 d (Table 1). This indicates that the most fertile cows were those that were inseminated during the first 90 d postpartum. This result is relevant because it contrasts with ^{Tixi et al. (2009}b), who obtained higher conception rates as time from calving elapsed. In this way, in the cited study the most fertile cows were those that were inseminated in their third and fourth services. In our study, the conception rate of cows with more than 150 d in milk was 14 % lower than that of cows in their first 90 d (22 % vs 36 %). The reason for this result could be that repeat-breeding cows are concentrated in the group inseminated after 150 d in milk. Thus, this group had already been selected because they suffered from diverse problems that cause fertilization failure and embryo death (^{Fourichon et al., 2000}; ^{Walsh et al., 2011}).

In our study, primiparous cows had a higher conception rate than multiparous cows (Table 1). The cause of higher fertility of primiparous cows is not clear. It may be related to higher production and dry matter intake of the multiparous cows, compared with primiparous cows (^{Berry et al, 2006}). Thus, multiparous cows would be more susceptible to the negative effects of subnormal concentrations of progesterone because of greater hepatic catabolism of steroid hormones (^{Sangsritavong etal, 2002}). In addition, it is possible that the multiparous cows are exposed to more factors that decrease fertility, such as dystocia, Puerperium pathologies and metabolic problems, than primiparous cows (^{Gröhn and Rajala-Schultz, 2000}; ^{López-Gatius et al, 2006}).

The Ovsynch protocol produced a higher conception rate than when insemination was performed at detected estrus (natural or PGF2a induced; Table 1). All the inseminations were performed after confirming genital signs of estrus, even in the Ovsynch protocol cows. In this context, the Ovsynch protocol cows may have been more fertile due to better synchronization between ovulation and insemination; cows in anestrus were not inseminated and the ovulatory follicle had fewer days of dominance (^{Pursley et al, 1995}).

The effect of the treatment on follicle dynamics was not measured in this study. However, the acute increase in blood progesterone concentrations causes atresia of the dominant follicle and emergence of a new follicular wave (^{Ahman et al, 1997}; ^{Diaz et al, 1998}). Thus, the injection of progesterone could have induced atresia of the dominant follicle on day five, early emergence of the second follicular wave and an estrous cycle with three follicular waves, and thus a longer luteum phase (^{Bergfeld et al, 1996}; ^{García et al, 2004}). In our study, the estrous cycle of progesterone treated cows was similar (p>0.10) to that of control cows (P₄ = 21.2±1.8 and control = 21.9 ± 1.6 days), suggesting that if there was an effect on follicular dynamics, it was not reflected in the duration of the luteum phase or the estrous cycle.

Another effect of modifying follicular dynamics following progesterone injection would be that the cows that did not conceive during the experimental cycle had a higher probability of conceiving in the following estrous cycle since, having three follicular waves, a follicle would be ovulating with fewer days of dominance. This would increase the potential of the oocyte to develop a viable embryo (^{Ahman et al, 1997}). However, the conception rate of the cows that returned to estrus within a normal interval (18 to 24 d) was 36 and 29 % (p = 0.31) for the P₄ groups and the control. In contrast, ^{Flores et al. (2013}) found that progesterone-treated cows under a similar protocol had a higher conception rate in the return to estrus than control cows.

After insemination, it is expected that all the cows that did not conceive would return to estrus within a normal period (18 to 24 d). However, some cows return in periods that are shorter (<17 d), longer (25 to 35 d), or double (36 a 48 d) (^{Tixi et al., 2009a}). Determining the intervals between services is a practical method to evaluate efficiency and precision in detecting estrus since it permits estimating the proportion of cows that were inseminated when they were not in estrus (short intervals) and the proportion of non-pregnant cows not detected when they returned to estrus. The main cause of long intervals is mainly the low efficiency in estrus detection. However, other factors may be involved, such as persistence of the corpus luteum and early embryo death (^{López-Gatius et al., 2002}; ^{Starbuck et al., 2004}). In our study, the distribution of intervals between services of the P₄ groups was similar to that of the control group (data not shown). The variation in conception rate obtained with cows returning to estrus when their intervals were different (Table 2) was notable. The cows with the highest conception rate were those that were inseminated after a normal interval, while the least fertile were those that were inseminated after a short or double interval. The causes of low fertility in these cows cannot be established with the observations made in this study.

Table 2 Return to estrus intervals after experimental insemination (data include cows from both groups). 

Conclusions

Administration of progesterone 5 days after insemination did not increase conception rate of dairy cows.