Conception rate in Holstein dairy cows having both normal sized follicles and cystic follicles at estrus

K. Kaneko

Laboratory of Veterinary Obstetrics and Gynecology, Azabu University, 1-17-71, Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan

Conception rate in Holstein dairy cows having both normal sized follicles and cystic follicles at estrus

K. Kaneko*

Laboratory of Veterinary Obstetrics and Gynecology, Azabu University, 1-17-71, Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan

ARTICLE INFO

Article history:

Received

Received in revised form

Accepted

Available online

Cattle

Cystic follicles

Normal sized follicles

Progesterone

Reproductive performance

Objective:To investigate the conception rate when cows showing clear signs of estrus and havingboth normal sized follicles and cystic follicles were artificially inseminated.Methods:Thirty parous Holstein cows showing clear signs of estrus and having both normal sized follicles (diameter ＜25 mm) and cystic follicles (diameter ≥25 mm) simultaneously in their ovaries were artificially inseminated.Changes in the ovaries were observed using real-time ultrasonography for 25 d after artificial insemination. The concentration of plasma progesterone (P4) was measured simultaneously to monitor the function of the corpus luteum.Results:The normal sized follicles ovulated in 21 out of 30 cows (ovulation rate: 70.0%), and 11 of these cows successfully conceived (conception rate: 36.7%) (Group C) while 10 cows did not conceive (Group NC). The maining 9 cows, normal sized follicles did notovulate (Group NO). The change in P4in cows that ovulated was similar to that in normal cows. Although there were no significant differences in median number and size of cystic follicles among the groups, all cows in Group C only had one cystic follicle.Conclusion:These results support artificially inseminated of cows that show clear signs of estrus even if they have both cystic follicles andnormal sized follicles, and especially when the cows have only one cystic follicle.

1. Introduction

Cows with cystic ovarian disease (COD) have abnormally large follicles (cystic follicles) with a diameter of at least 25 mm that persist for more than 10 d. These cystic follicles are steroidogenic and secrete estradiol or progesterone. As a result, they interfere with normal ovarian cyclicity[1-3]. Cows with COD are generally infertile as long as the cystic follicles remain steroidogenic[3]. However, COD can resolve spontaneously[4-6], and somecystic follicles become non-steroidogenic even if the structures remain[3,7,8].

Although non-steroidogenic cystic follicles are hormonally inactive and do not influence the normal estrous cycle[3,9-13], it is impossible to determine whether the cystic follicles are steroidogenic or nonsteroidogenic by rectal examination or real-time ultrasonography. In the non-steroidogeniccase, estrus occurs even if cystic follicles are present[1]. It is therefore possible that normal follicles and nonsteroidogenic cystic follicles coexist, and cows may be able to conceive if they areinseminated before the normal follicles ovulate. However, there are no reports about the reproductive performance of cows having both normal follicles and cystic follicles. Cows that are confirmed to have cystic follicles by rectal examination or real-time ultrasonography will be diagnosed as having COD, and are normally given hormonal treatment but are not artificially inseminated[14]. As a result, these cows, which have non-steroidogenic cystic follicles, have extended calving intervals[2,15-17]. The most important parameter for determining the economic losses due to COD is the sum of days from calving to insemination[18]. The primary objective of this study was, therefore, to investigate the conception rate whencows showing clear signs of estrus and havingboth normal sized follicles and cystic follicles were artificially inseminated. Ovarian dynamics and blood progesterone concentration (P4) after artificial insemination (AI) were also monitored.

2. Material and methods

2.1. Animals

Cows showing clear signs of estrus and found to have both normal sized follicles and cystic follicles in their ovaries by rectal examination were selected for this study. In accordance with the definition of COD[2], follicles with a diameter ＜25 mm were considered normal sized follicles, and those with a diameter ≥25 mm were considered cystic follicles. Thirty parous Holstein cows between 2 and 6 years of age were selected. Although the cows were kept at 9 different farms, they were all housed in a free stall barn, fed a total mixed ration and had free access to water. Their body conditioning score was 2.5–4.0, and their milk volume was 25 to 40 kg/d. The signs of estrus included sniffing the genitalia of other animals, discharge of cohesive strands of mucus from the vulva, increased bellowing, mounting, standing, and chin resting. The cows were artificially inseminated when they showed signs of estrus, and this was considered day 0. Cows with a history of treatment for ovarian or uterineabnormalities (COD, persistent ovarian follicles, endometritis, and pyometra) were excluded from this study.

2.2. Ultrasound scanning

Ovaries were examined on days 0, 1, 10, and 20 using an ultrasonograph (Model SSD-500; Aloka, Tokyo, Japan) equipped with a 5-MHz transrectal linear transducer. The presence of normal sized follicles was confirmed on day 0, and the number of cystic follicles was counted. The size of follicles was expressed as an average of a minor axis and the major axis. Ovulation of normal sized follicles was checked on day 1, and ovarian dynamics were monitored on days 10 and 20. Pregnancy was confirmed at 30 d after AI by ultrasonography.

2.3. Blood sampling

A vacuum-type heparinized tube was used to collect blood samples from the tail vein on days 0, 5, 10, 15, 20, and 25. Plasma was separated by centrifugation (2000×g for 10 min) and stored at –80℃ until the determination of P4.

2.4. Hormone analysis

P4was measured using acommercial radioimmunoassay kit for progesterone (Diagnostic Products, Los Angeles, CA, USA), which has been previously validated for cattle[19]. Intra- and inter-assay coefficients of variation were 8.8% and 9.7%, respectively, and the average sensitivity of the assay was 0.02 ng/mL.

2.4. Statistical analysis

The cows were assigned to three groups: Group C (normal sized follicles ovulated on day 1, and cows successfully conceived), Group NC (normal sized follicles ovulated on day 1; however, cows did not conceive), and Group NO (normal sized follicles did not ovulate). Among the groups, the number and size of cystic follicles were analyzed using a Mann–Whitney U test. Results are reported as mean±SD when normally distributed and as median (minimum–maximum) when skewed. The fate of cystic follicles was analyzed using a Chi-square test followed by Fisher's exact test. The P4data were analyzed using repeated measures analysis of variance (treatment time) followed by Fisher’s protected least significant difference test.P＜0.05 were considered statistically significant difference.

3. Results

3.1. Ovulation of normal sized follicles and conception rate

Ovulation of normal sized follicles occurred in 21 of 30 cows (70.0%) on day 1, and 11 of these cows (36.7%) successfully conceived (Group C), while 10 cows did not conceive (Group NC). None of the nine cows with normal sized follicles that did not ovulate on day 1 (Group NO) conceived Table 1 and Table 2.

Table 1The state of cystic follicles.

Table 2The fate of cystic follicles.

3.2. Change in P4

The change in P4in the 11 cows in Group C was similar to that in cows with normal estrus cycle[20]. P4increased considerably after AI, reached ＞1 ng/mL on day 5, and remained at this level throughout the observation period. P4increased to ＞1 ng/mL on day 5 and remained at this level until day 15 in cows in Group NC, but it decreased on day 20 and dropped to 1 ng/mL on day 25. P4did not increase in cows in Group NO and remained approximately 1 ng/mL throughout the entire observation period (Figure 1).

Figure 1.Mean ± SEM plasma P4concentration between day 1 and day 25 in each group: Group C (●) (n=11); Group NC (▲) (n=10); Group NO (■) (n=9).

3.3. Number and size of cystic follicles

The median number (minimum–maximum) of cystic follicles was 1 (1–1) in Group C, 1.8 (1–3) in Group NC, and 1.6 (1–2) in Group NO. The number of cows with multiple cystic follicles was 0 (0%) in Group C, 4 (40.0%) in Group NC, and 4 (44.4%) in Group NO. The median cystic follicle size was 34.9 (27–50) mm in Group C, 30.6 (25–45) mm in Group NC, and 36.1 (25–50) mm in Group NO. There were no significant differences in these parameters among the groups.

3.4. Ovarian dynamics

A corpus luteum (CL) developed after ovulation of normal sized follicles in all cows in Group C and Group NC even when cystic follicles were present (Figure 2). However, all CL of cows in Group NC had regressed by approximately Day 20. There were three outcomes for cystic follicles present on Day 0. They were maintained during the observation period (10 in Group C, 15 in Group NC, and 13 in Group NO), luteinized (one in Group NC), or they regressed and disappeared (1 in Group C, 2 in Group NC, and 1 in Group NO). New cystic follicles also developed during the observation period (3 in Group NC and 2 in Group NO). However, there were no significant differences intheseparametersamong the groups.

Figure 2.Ovarian dynamics of one cow in Group C. The normal size follicle in the left ovary on day 0 ovulated and the corpus luteum developed on day 10, and it was maintained until day 20. In the right ovary, the cystic follicle continued to exist during the observation period. Scale bars, 10 mm.

4. Discussion

In this study, ovulation of normal sized follicles occurred in 70.0% of all cows, and the conception rate was 36.7%. Furthermore, although 10 cows did not conceive after ovulation of normal sized follicles, CL developed and secreted P4successfully. This confirmed previous reports that some normal sized follicles that coexist with cystic follicles can ovulate normally[9,12]. Cows with COD recover spontaneously in some cases[4-6], and steroidogenic cystic follicles become non-steroidogenic during this recovery[3,7,8]. Nonsteroidogenic cystic follicles are hormonally inactive and do not influence the normal estrous cycle[3,9-13]. In these cases, the ovaries have normal function and cyclicity, and normal estrus occurs even when cystic follicles are present[1].

In this study, cystic follicles of cows in Group C and Group NC were considered to be non-steroidogenic, because normal sized follicles ovulated and CL developed. On the other hand, normal sized follicles of cows in Group NO did not ovulate, and there was no significant increase in P4during the observation period. Furthermore, new cystic follicles developed in two cows. These results suggest that thecystic follicles of cows in Group NO might have been steroidogenic. Moreover, three new cystic folliclesdeveloped in cows in Group NC after ovulation of normal sized follicles. This indicates that endocrinological abnormalities might have also persisted in some of the cows in Group NC.

It is essential to differentiate between steroidogenic and nonsteroidogenic cystic follicles when cows show clear signs of estrus and have both normal sized and cystic follicles. The median number and size of cystic follicles did not differ among the groups in this study. Consequently, the number and size of cystic follicles could not be used as indicators of whether cystic follicles were steroidogenic or non-steroidogenic. Even cows with steroidogenic cystic follicles exhibit periods of follicular growth that are associated with increased estradiol secretion[9,21]. Therefore, it is difficult to determine whether cystic follicles are steroidogenic or non-steroidogenic. However, all cows in Group C had only one cystic follicle. This might suggest that normal sized follicles are normal pre-ovulatory follicles when there is only one coexisting cystic follicle.

Ovulation of normal sized follicles occurred in two-thirds of cows in the present study, and the conception rate was 36.7%. This rate is close to the reported conception rate of 34.0% for cowsinJapan after the first AI[22]. This suggests that to shorten the parturition interval it is worth artificially in seminating cows that show clear signs of estrus even ifthe cows have both cystic follicles andnormal sized follicles. Because a gonadotropin-releasing hormone analogue induces a surge of luteinizing hormone that is similar to the preovulation surge[23], prescribing gonadotropin-releasing hormone to cows that show signs of estrus and have both normal sized and cystic follicles at AI may be recommended for inducing ovulation of normal sized follicles and for treatment of COD.

Conflict of interest statement

The authors declare that they have no conflict of interest.

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10.1016/j.apjr.2016.07.012

*Corresponding author: Kazuyuki Kaneko, Azabu University1-17-71, Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan.

E-mail: kaneko@azabu-u.ac.jp