Introduction

The low efficiency of estrus detection in dairy herds, has motivated the development of artificial insemination (AI) programs without the need to detect cows in estrus. The first program validated with these characteristics is known as Ovsynch and has served as the basis for other schemes. This program begins with an injection of GnRH in early diestrus (day zero), followed by injection of PGF2α (d 7); subsequently administration of the second GnRH dose (d 9) and insemination occurs 16 h later. The first injection of GnRH induces ovulation or luteinization of ≥ 8 mm diameter follicles, which synchronizes the emergence of a follicular wave, so at the moment of the PGF2α injection cows continue on diestrus and have a preovulatory follicle in the same state of development; with the second injection of GnRH ovulation is induced, which is synchronized with the fixed-time artificial insemination¹. The proportion of pregnant cows of the total inseminated [pregnancy rate (PR)] obtained in this program is similar to that attained when cows are inseminated at observed estrus, either natural or synchronized with PGF2α ^1,2. However, the advantage of the Ovsynch program is that all cows that are enrolled in the program can be inseminated, while in programs of insemination at observed estrus only those detected in estrus are inseminated (between 40 and 60 % from the total eligible)^1,3.

Although the coherent physiological concept of the Ovsynch program, results in dairy herds are not the optimum. Among the most important factors that determine the results are: the stage of the estrous cycle in which the synchronization of the follicular wave starts⁴, the characteristics of the ovulatory follicle^5,6, the cyclicity of the cows enrolled in the program⁷, the progesterone concentrations during the program^8,9, as well as management factors, which determine that not all cows that start the program complete it correctly².

Within the modifications that have been made to optimize the basic Ovsynch protocol it stresses the inclusion of progesterone^10-12. The combination with progesterone has favored the PR up by 10 percentage points⁸, which is related to a better environment for the ovulatory follicle development¹³; in addition, the incorporation of progesterone can induce a fertile estrus in females that are in anoestrus or in the transition phase of the anoestrus to cyclicity¹⁴. On the other hand, by shortening the time between the first injection of GnRH and the administration of the PGF2α has been a decrease of the period of dominance of the ovulatory follicle. So, a program was developed in which cows are inserted with a progesterone-releasing intravaginal device for 5 d. The day of insertion they receive the first injection of GnRH and upon removing the device are injected PGF2α, repeated 24 h later. The second injection of GnRH is at 56 h after removal of the device and cows are inseminated 16 h later^10-12.

According to this information, the program with progesterone would generate better results than basic Ovsynch protocol; however, there is no sufficient field studies with dairy cows in which such programs can be contrasted. Therefore, the objective of the present study was to compare the PR between cows inseminated by the common Ovsynch and cows inseminated with an Ovsynch program, in which cows receive progesterone for 5 d and also the period between injection of GnRH and PGF2α is reduced from 7 to 5 d.

Materials and methods

Design of the experiment

Two hundred and seventy five (275) cows of different parity and body condition score greater than 2 (scale of 1 to 5) were pre-synchronized with two PGF2α i.m. injections (Enzaprost^®, Ceva Animal Health, Mexico) 14 d apart, at 32 ± 3 d postpartum (DPP). Twelve (12) days after the second PGF2α injection (58 ± 3 DPP), cows were randomly assigned to the following treatments: 1) Group Ovsynch (n=144), received a GnRH i.m injection at 0800 (Cevarelin ®, Ceva Animal Health, Mexico), 7 d later the cows received a PGF2α i.m. injection (Enzaprost^®) (65 ± 3 DPP; 0800); 56 h later received a second injection of GnRH (67 ± 3 DPP; 1600) and were fixed-time inseminated 16 h later (68 ± 3 DPP; 0800). 2) Group PRID5d (n=131) received an i.m. GnRH injection at 0800 and a releasing intravaginal progesterone device (PRID Delta ®, Ceva Animal Health, Mexico), which remained in situ for 5 d; upon removing the device (63 ± 3 DPP; 0800) PGF2 α injection was applied followed by the second PGF2α 24 h later; the second dose of GnRH was applied 56 h after removing the device (65 ± 3 DPP 1600) and cows were inseminated at fixed-time 16 h later (66 ± 3 DPP; 0800). After the second injection of PGF2α during the presynchronization period, all the cows were fitted with heat mount detectors patch for estrus detection and the proportion of cows showing estrus was determined. Pregnancy diagnosis was performed by rectal palpation between 45 and 50 d post-insemination.

Figure 1 shows the two treatments in a schematic way. It should be noted that in the present study two different insemination timefixed protocols were compared: group PRID5d is different from the Ovsynch group because the first includes 5 d of progesterone and the reduction of 2 d in the period between injection of GnRH and PGF2α (from 7 to 5 d). This clarification is relevant, because having difference between the groups, it could not be established whether due to the inclusion of progesterone or the modification of the interval between the injection of GnRH and PGF2α.

Figure 1 Schematic overview of the treatments. 

Results

The PR was similar between treatments (27 vs 21 %; Ovsynch and PRID5d, respectively). There was no effect on PR from the milk production, parity, AI technician, or the type of puerperium (Table 1). The interaction of the treatment with these variables did not affect PR (P>0.1). The proportion of cows showing estrus after PGF2α injection prior to the start of the synchronization of ovulation (first injection of GnRH) was similar between treatments [77 % (111/144) vs 78 % (102/131); Ovsynch and PRID5d, respectively]. The presentation of the estrus prior to the first injection of GnRH did not affect PR and the interaction between treatments with this variable did not affect PR (P>0.1). The PR was similar between the cows that did not show estrus [Ovsynch (12.1 %; 4/33) vs PRID5d (20.7 %; 6/29). P=0.36].

Table 1 Odds ratio for pregnancy probability according to the treatment and some productive and reproductive variables in dairy cows*. 

*There was not interaction (P>0.1) among the treatments and analyzed variables on pregnancy rates (PR).

Discussion

In the present study it was expected that the inclusion of progesterone in the PRID5d group would improve PR; however, the result was similar to that obtained with basic Ovsynch program. The global PR accomplished in both treatments are equal to that obtained in similar synchronization programs¹⁷ and also coincides with that obtained by insemination at observed estrus^18,19.

It was expected that the cows in the PRID5d protocol had higher PR than cows that did not receive progesterone, as experimental evidence so indicated it⁸. In several studies, the administration of progesterone during the follicular wave timing and throughout ovulatory follicle maturation has improved PR^10,20,21. In addition, cows that are cycling and have a corpus luteum (CL) at the time of the first injection of GnRH have PR greater than cows in anestrus^7,22; however, in the present study the cows that received progesterone showed similar PR. The cause of this result is unknown, but some possibilities are exposed in the following paragraphs.

The time of dominance of the ovulatory follicle in Ovsynch programs affects the PR. The ovulation of very small follicles, i.e. follicles that reach the dominance very near the time of the induction of luteal regression, can produce low estradiol concentrations and increase premature regression of the CL of the subsequent estrus cycle (short luteal phase) and loss of pregnancies^23-25. On the other hand, the ovulation of follicles with long periods of dominance (too large follicles) also affect the PR, possibly because released oocytes are less competent to develop viable embryos^26-28. In protocols of fixed-time insemination in beef cattle^10,25,29, the reduction of the period of dominance of the follicle ovulation through the decrease of 2 d between the first injection of GnRH and the administration of PGF2α (7 vs 5 d) in conjunction with the elongation of the proestrus resulted in best PR. The absence of improvement of the PR observed in this study probably lies in that the period between the administration of PGF2α and the induction of ovulation with the second injection of GnRH was 56 h while in the cited studies it was 72 h. This temporary difference may have influenced the characteristics of the ovulatory follicle, giving as a result an ovulation of a small follicle, which is associated with PR reduction^25,30.

On the other hand, a disadvantage of reducing the period between the first injection of GnRH and PGF2α (5 d) administration, is that the CL after ovulation induced with GnRH is resistant to the effect of PGF2α ^11,12,31. Even in the basic program of Ovsynch whereby after 7 d between GnRH and PGF2α, failure in luteolysis in a range of 5 to 30 % has been observed (incomplete luteolysis)^6,32, causing cows to present suprabasal concentrations of progesterone at the time of insemination. Thus, progesterone concentrations above 0.3 to 0.5 ng/mL at the time of insemination are associated with low pregnancy rates³². Treatment with two injections of PGF2α with difference of 8 h, or a single injection with a double dose of PGF2α in cows with a CL (with the same days as in the present study) has been evaluated, and in both cases the luteolysis was incomplete³¹. Although in the present study the cows from the PRID5d group received two injections of PGF2α 24 h apart, it cannot be ruled out that a luteolysis failure has prevented the cows of this group to show a PR increase.

It is also possible, that the absence of a positive effect of the progesterone is because this hormone supplementation favors PR, especially in cows that have not yet started postpartum ovarian activity^2,33. In the present study, the proportion of cows showing estrus after PGF2α injection prior to timed artificial insemination program was similar between groups (77 vs 78 %; Ovsynch and PRID5d, respectively), which indicates that a high proportion of them was cycling and would have, hypothetically, a CL at the beginning of the Ovsynch program. In these circumstances, the effect expected for by the addition of progesterone in the synchronized protocol might be irrelevant. It should be noted that in timed artificial insemination programs in anestric beefs cows, the inclusion of progesterone enhances PR in both, Bos taurus¹⁰ and Bos indicus³⁴.

The PR in both treatments is consistent with that obtained in similar synchronization programs^17,33 or when cows are inseminated after estrous detection^18,19. However, the proportion of pregnant cows of the total eligible to inseminate (pregnancy rate) was twice in the programs evaluated in comparison with the that obtained in contemporary inseminated cows when they were detected in estrus (spontaneous or induced) with PGF2α. In the present study, 40 % of the contemporary cows eligible to inseminate were detected in estrus and were served, while 100 % of cows in the evaluated protocols were inseminated. With these data, the pregnancy rate in the experimental groups was 24 % vs 12 % in contemporary cows (this was estimated considering PR of 30 % and 40 % efficiency in estrus detection). It is noteworthy that the pregnancy rate in Mexico varies from 12 to 15 % and is similar to that observed in the United States^2,3. This difference in the pregnancy rate could economically justify the fixed-time insemination programs. In a study done in Mexico in 2011³⁵, using Monte Carlo methodology³⁶ estimated the economic consequence of changes in the pregnancy rate and found that for every percentage point that it increases in the range of 15 to 20 %, generates an additional income of $748.00 Mexican pesos per cow per year.

Something that should be highlighted in this study is that milk production did not affect the PR. In the past 30 yr there has been an association between the increase in milk production and the decrease of the PR³⁷, although this is not necessarily true. Lopez-Gatius et al³⁸ found that cows that produced more milk (>50 kg) on d 50 postpartum were most fertile. Although a result similar to the above³⁸ was not found in the present study, neither there was not a decrease in the PR in cows that produced more than 40 kg of milk compared to those that produced less than 40 kg.

In the present study, primiparous cows had similar PR to multiparous (31 vs 20 %, respectively, P=0.21), which contrasts with other studies^19,38, probably by the few number of observations.

The type of puerperium did not affect the PR, which is different to that found in other studies, in which the cows that had any uterine postpartum pathology were less fertile in the first service than cows with a normal puerperium³. The cause of this result is unknown, but it can be speculated that it is related to the clinical management of the cows with abnormal puerperium, since in addition to receiving the respective drug treatments, they were subjected to systematic PGF2α injection every 14 d from the d 32 post-partum (presincronization). Thus, luteolysis and estrus favored the removal of uterine infections and could avoid its negative effect on the PR of the first service³⁹.

Conclusions and implications

It is concluded that the PR was similar among the cows inseminated with a common Ovsynch program or by using an Ovsynch program which includes 5 d of progesterone and the reduction of 7 to 5 d between injection of GnRH and PGF2α.