THE EFFECT OF THE INTENSITY OF ESTRUS EXPRESSION ON THE FOLLICULAR DIAMETER AND FERTILITY OF NELLORE COWS MANAGED

The aim of this study was to determine the relationship between the intensity of estrus expression on the ovulatory follicle diameter (OFD) and its effect on the conception rate in zebu females managed under a FTAI program. On a random day of the estrous cycle, day 0, 308 Nellore females received an intravaginal progesterone device and 2.0 mg of estradiol benzoate were administered intramuscularly (IM). On day 8, the progesterone device was removed, and 300 IU of equine chorionic gonadotropin, 150 μg of d-cloprostenol, and 1.0 mg of estradiol cypionate were administered IM. The animals were then painted with animal marker spray between the sacral tuberosity and the tailhead. On day 10, the animals were categorized into three groups: no estrus expression (n = 78), low intensity of estrus expression (n = 66) and high intensity of estrus expression (n = 164). The OFD was then measured, and artificial insemination (AI) was performed. The results suggest that determining the intensity of estrus expression is a good parameter to identify females with greater follicular diameter and increased fertility in Nellore cows submitted to a FTAI protocol.


Introduction
The reproductive efficiency in beef cattle remains characterized by low production and conception rate in spite of the advancement in animal reproduction biotechnologies.Therefore, improved herd genetics and increased production efficiency are necessary to increase the economic return of the beef cattle industry in Brazil (1) .
Among the available biotechnologies, artificial insemination (AI) is an important tool to promote genetic improvement and to increase the reproductive and production efficiency of beef cattle (2) .However, postpartum anestrus, delayed puberty, and the failure to detect estrus are the main factors that limit the reproductive success of AI programs in Bos taurus indicus (zebu) herds (3) .The low efficiency of estrus detection in zebu females is because of the reproductive behavior characteristics of these females, which have a shorter estrus that manifests at night, thereby making it difficult to use this biotechnology in beef cattle herds (4,5) .Thus, various hormonal treatments have been developed to manipulate follicular dynamics and synchronize estrus and ovulation cycles, which enable the use of fixed-time artificial insemination (FTAI) without detecting estrus in cows (6) .Despite the evolution of hormonal protocols and the wide use of FTAI, the results must be improved (7) .
Studies have been conducted to improve the reproductive and success rates of FTAI programs in beef cows (8) .Ovulatory follicle diameter (OFD) is an important indicator of fertility in zebu females subjected to the synchronization protocol of FTAI (9) .However, measuring the follicular diameter at the time of AI requires advanced technology, such as an ultrasound machine and a trained technician (10,11) .
Recent studies have shown that the presence of an ovulatory follicle at FTAI in females subjected to a synchronization protocol is related to estrus, increased ovulatory capacity, and a higher FERRAZ, P.A. et al.
probability of conception (12) .Females in estrus have higher concentrations of estradiol at AI and hence a higher likelihood of conception compared to females that are not in estrus (13) .
Recently, auxiliary methods have been developed to increase the efficiency of estrus detection in cows.In this context, animal marker spray can be used as a practical and accessible tool for estrus detection between the removal of the progesterone source (P4) and FTAI, and it is an alternative for improving the efficiency of synchronization protocols for FTAI, thus replacing the more costly methods that are difficult to apply in beef cattle herds (14) .
The aim of this study was to evaluate the relationship between the intensity of estrus expression, OFD, and conception rates of zebu females subjected to a synchronization protocol for FTAI using the animal marker spray method to identify the intensity of estrus expression.

Materials and Methods
The experiment was conducted between September and December 2012 on the API Ranch located in the municipality of Catu at the Recôncavo region of Bahia, Brazil.The region has an average annual rainfall of 1,400 mm and has a humid tropical climate.
In total, 308 Nellore females (Bos taurus indicus) were included in the study.These animals belonged to the category of parous cows with a mean age of 4.07 ± 2.34 years, body condition score of 3.19 ± 0.42 (scale from 1 to 5) (15) , and postpartum interval of 64.58 ± 18.42 days.The animals were maintained on pasture predominantly consisting of Brachiaria decumbens with mineral supplementation and water ad libitum.
Prior to the implementation of the FTAI protocol, the females received a clinical gynecological examination and transrectal ultrasound with a linear transducer at a frequency of 5.0 MHz (Mindray, DP2200vet, São Paulo, Brazil).The cows without reproductive tract abnormalities at the evaluation were considered suitable participants for the experiments.
The females were subjected to the following synchronization protocol: on a random day of the estrous cycle, day 0 (D0), parous cows received a new intravaginal device containing 1.9 g of progesterone (P4, CIDR ® , Pfizer, São Paulo, Brazil).All cows received 2.0 mg of estradiol benzoate (GONADIOL ® , Tecnopec, São Paulo, Brazil) intramuscularly (IM) with the insertion of the progesterone-releasing intravaginal device.On day 8, the progesterone-releasing intravaginal device was removed, and 300 IU of equine chorionic gonadotropin (eCG, NOVORMON ® , MSD Saúde Animal, São Paulo, Brazil), 150 µg of d-cloprostenol (PROLISE ® , Tecnopec, São Paulo, Brazil), and 1.0 mg of estradiol cypionate (ECP ® , Pfizer, São Paulo, Brazil) were administered IM.The cows were painted with color chalk (RAIDEX ® , GmbH, Dettingen/Erms, Germany) between the sacral tuberosity and the tailhead.The females remained in the same paddock so that the females in estrus could accept mounting but there was only female-to-female interaction, which was noted by the removal of the animal tail chalk.
On day 10, the females were classified into three groups based on estrus expression: the tail chalk that was not removed (n = 78) characterizing the non-estrus group; the tail chalk that was partially removed (n = 66) characterizing the group expressing low intensity of estrus; and the tail chalk that was completely removed (n = 164) characterizing the group expressing high intensity of estrus.
The effect of the intensity of estrus expression on the folicular diameter and fertility... Furthermore, the OFD was measured by transrectal ultrasound using a linear transducer at a frequency of 5.0 MHz (Mindray, DP2200vet, São Paulo, Brazil).Both ovaries were examined, and the diameter of the largest follicle at this time was identified and measured.The animals were inseminated 48 to 54 h after removing the progesterone device with doses of cryopreserved semen from five Nellore bulls with known fertility, thawed at 37 °C for 30 seconds, and distributed homogeneously among the experimental groups.The sperm parameters of the semen doses were within the minimum values established by the Brazilian College of Animal Reproduction (16) .In this study, to avoid effects of the insemination technician, the same technician performed all the inseminations.
Pregnancy was diagnosed using a transrectal ultrasound at 35 days post-insemination.The presence of an embryonic vesicle with a viable embryo (heartbeat) was considered a positive pregnancy diagnosis.The conception rate was calculated by dividing the total number of pregnant cows by the number of inseminated cows.All animal interventions began at 08:00 a.m.Data were analyzed using the Statistical Package for Social Sciences (SPSS version 19), considering a significance level of 5%.The variables studied were ovulatory follicle diameter, intensity of oestrus expression, and response to pregnancy diagnosis after insemination.Therefore, the following sequence of tests was carried out: comparison between experimental groups regarding DFOL and intensity of oestrus expression was performed by Mann-Whitney test due to nonnormality.The conception rate between the experimental groups was compared by applying a frequency dispersion study using the chi-square test (χ 2 ).
As for the response variable, pregnancy diagnosis was assumed to present binomial distribution and it was analyzed based on logistic regression methodology.The objective of this analysis was to verify the significance of each variable in explaining the variation in the diagnosis of pregnancy, to quantify the change in the chance of pregnancy for each unit of increase in regression variables and use these variables to predict the probability of pregnancy (Pi).The tested variables were intensity of estrus expression and diameter of the ovulatory follicle.The model adjusted by stepwise method for probability of pregnancy (Pi) was: where yij = -2.25 + 0.25ESTRUS1+0.79ESTRUS2+0.14DFOL, Yij = probability of pregnancy, ESTRUS1 = expressing low intensity estrus, ESTRUS2= expressing high intensity estrus, OFD = Ovulatory follicle diameter.The 2 R max (which can reach statistical maximum value of 1) of the model was 12%.

Results and Discussion
In this study, the intensity of estrus expression influenced OFD.Comparisons between the group without estrus expression and low intensity of estrus expression showed no significant difference, but the group with a high intensity of estrus expression had a larger OFD (p = 0.0002) than the other groups.The average sizes of the OFD among groups are described on Table 1.
In the present study, the intensity of estrus expression between the removal of the progesterone device and the time of FTAI was an important indicator of increased follicular diameter in Nellore females subjected to a protocol to synchronize ovulation.The results were similar to those reported by other studies on zebu cows (8,17,18) .Larger ovulatory follicles at the time of AI have been associated with elevated concentrations of estradiol and consequently a high incidence of estrus expression (19) .Similarly, Atkins et al. (20) reported a significant positive correlation between estradiol concentration and follicular diameter.
Based on the results, there were significant differences in the conception rate between the different experimental groups: no estrus expression (32.1%), low intensity of estrus expression (37.9%), and high intensity of estrus expression (62.2%).Comparisons of the conception rates between the groups with no estrus expression and a low intensity of estrus expression showed no significant differences, but the group with a high intensity of estrus expression had a higher conception rate (p=0.0001)than the other groups (Table 2).
Therefore, the positive association between the likelihood of conception and estrus expression is potentially related to higher concentrations of estradiol in females expressing estrus (21) .These high levels of estradiol at insemination seem to favor fertilization by influencing sperm transport (22,23) and promoting positive changes in the uterine environment (24) .Under the influence of estrogen, a decrease in uterine pH is observed.This change in pH resembles that of seminal plasma regarding pH (25) .Thus, the reduction in uterine pH leads to a decrease in sperm motility and metabolism, which increases its viability and permanence in the female reproductive tract until ovulation, thus favoring fertilization (26) .
According to Perry et al. (11) , this improvement in the conception rate of females expressing estrus might have occurred because these females had a higher ovulation rate and better synchronization of ovulation compared to females that did not express estrus.This phenomenon was triggered by the size of the ovulatory follicle at the time of FTAI and by high concentrations of estradiol, promoting a higher rate of conception.Furthermore, Sá Filho et al. (17) reported that estrus following the synchronization protocol can improve the efficiency of FTAI protocols in Nellore cows and can be used as a strategy to improve the use of sexed semen in reproductive programs.
In this study, based on the conception probability according to OFD, it was also possible to estimate the probability of conception for the regressor variable OFD, as seen in Figure 1.These results are similar to those reported by Ribeiro Filho et al. (13) , who observed that an increase in OFD led to an increased likelihood of conception.In Nellore females who had OFDs larger than 13.60 mm, the mean estimated likelihood of conception was 78.83%, which was significantly higher than that in females with diameters of up to 11.20 mm and between 11.20 and 13.60 mm, 33.78 and 59.17%, respectively.Similarly, Borsato et al. (28) studied Bos taurus taurus X Bos taurus indicus heifers and observed a linear increase between conception rate and OFD.These authors observed that animals with ovulatory follicles between 7 and 10 mm had conception rates of 11.1%, whereas those with 10-to 13-mm follicles had a conception rate of 63.8%, and those with 13-to 19-mm follicles had a conception rate of 88.2%.
Females with larger follicles have a higher ovulation rate, a larger corpus luteum diameter and, consequently, a higher concentration of progesterone compared to females that have smaller follicles (12) .In addition, Vasconcelos et al. (29) reported that the lower progesterone level from females with smaller follicles, can affect the fertility because of insufficient embryonic development and reduced IFN-tau production by the embryo.After ovulation, progesterone plays a fundamental role in endometrial secretion, recognition and maintenance of pregnancy, and embryonic development (30) .
Additionally, the logistic regression indicated that on average, females with high and low intensity estrus expression had a 56 and 39% probability of becoming pregnant, respectively, versus 31% for females with no estrus expression (Figure 2).