The period between calving and first insemination is referred to as Voluntary Waiting Period (VWP)^4,5. Maintaining a 12-month calving interval in dairy cows requires the animal to conceive again as soon as possible after calving. To reach such a calving interval, uterine involution and re-establishment of endocrine function must be completed during the VWP^6,7. The VWP should be preferably around 45-50 days^8,9. In Holstein cows, the first wave of follicular growth begins around two weeks after calving about In about 30% to 40% of these cows, ovulation occurs ovulation occurs between days 16 and 20 pp in about 30% to 40% of these cows. However, 30% to 40% of cows ovulate between 30 and 50 days pp, while about 20% to 40% remain anovulatory until 50 to 60 days pp^10-12.

An early return of ovarian cyclicity improves reproductive efficiency, leading to greater milk yield¹³. In contrast, delayed ovarian cyclicity during the pp period negatively affects reproductive performance¹⁴. The onset of ovarian cyclicity, the first ovulation, and the continuation of regular estrous and ovulatory cycles after calving are influenced by several factors, including breed, parity, season, body condition score (BCS), postpartum diseases, and nutrition¹⁵.

Although extended VWP is known to impair fertility, there isn’t enough research on this topic in Holstein cows raised under Turkish conditions. To adress this gap, this study aimed to evaluate certain biochemical parameters that may influence the length of the VWP in Holstein cows and to investigate the effects of an extended VWP on some reproductive parameters.

Animals, Nutrition and Management Conditions: The study included 30 healthy Holstein cows aged 2-6 years from a private dairy farm in Tekirdağ Province, Türkiye. All the cows had recently given birth and had no history of puerperal illnesses such as dystocia, retained placenta, endometritis, laminitis, or displaced abomasum. All animals received the same care, food, and reproductive management, and were fed ad libitum twice a day with total mixed rations designed for the transition period. Clean drinking water was continuously available to the animals. All cows included in the study were with normal BCS values.

Study Design: All cows in the study were examined twice, on postpartum (pp) days 45 and 55, by rectal and ultrasonographic examination (Hasvet WED-3100V) at a 10-day intervals. Cows that had a corpus luteum (CL) detected on at least one of these examinations and a blood progesterone level >1 ng/mL were classified as cyclic (n= 15), while cows with no CL detected in either examination and a progesterone level <1 ng/mL were considered acyclic (n= 15). On the day 55 pp, which is the day of the second examination, blood samples were collected from the tail vein of all animals. The age and BCS of each animal were recorded. The blood samples were centrifuged at 3000 rpm for 15 minutes, and the serum was separated and stored at –80°C until analysis. β-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), glucose, cholesterol, and triglyceride levels were measured using an autoanalyzer (Randox, RX Imola, Crumlin, United Kingdom). Progesterone was determined using the chemiluminescence technique with an autoanalyzer (Roche Cobas e 701). The first estrus following the VWP was determined based on increased activity measured by pedometer, increased uterine tone on rectal palpation, and the presence of a Graafian follicle in the ovaries. Reproductive performance of the animals were evaluated using the farm records to determine the number of days from calving to first insemination and from calving to conception for each animal included in the study.

Statistical Analysis: Normality of the data was assessed using the Shapiro-Wilk test. Parameters showing normal distribution were analyzed using the parametric unpaired t-test, while those not normally distributed were analyzed with the non-parametric Mann-Whitney test. All analyses were conducted using the GraphPad Prism 9.4.1 software. Statistical significance was set at p<0.05.

In acyclic cows, the intervals from calving to first estrus, calving to first insemination, and calving to conception were found to be significantly shorter compared to cyclic cows (p<0.01) (Table 2).

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Table 1: Distribution of histopathological parameters in the groups

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Table 2: Comparison of reproductive parameters between the groups

Changes in BCS during the transition period are regarded as markers of negative energy balance (NEB)¹⁶. Cows that resume cyclic activity early after calving have been reported to have higher BCS compared to acyclic cows. The reason for this is that cows with a higher BCS usually have better nutritional condition and sufficient gonadotropin secretion^17,18. However, the present study found no significant difference in BCS between cyclic and acyclic cows (p>0.05). The similarity in nutrition and management strategies between both groups could explain why this difference did not reach statistical significance.

It has been reported that parity has a significant effect on the resumption of ovarian activity after calving. Cows that gave birth for the first time typically experience a later onset of ovarian cyclicity compared to those that calved multiple times^19-21. However, in the present study, no significant difference was found between the parities of cyclic and acyclic cows (p>0.05). The reason for this could be the fact that the animals in the study were not grouped as primiparous and multiparous and were selected with similar parities.

Non-esterified fatty acids and BHB are considered key indicators of NEB. It has been reported that there is a relationship between the resumption of ovarian cyclicity and NEB after calving²². Elevated levels of NEFA and BHB, which are indicators of NEB, have been shown to negatively affect ovarian activity^23,24. However, other studies have also reported that NEFA and BHB do not directly affect the resumption of ovarian cyclic activity²⁵. In the present study, no significant difference was found between the NEFA and BHB levels in cyclic and acyclic cows (p>0.05). These results are consistent with the findings of Teixeira et al.²⁵. The lack of disparities between the groups suggests that both were controlled with efficient feeding regimens. Differences between studies could be attributed to the number of animals in each group and the feeding regimens they followed.

The effects of blood glucose levels on postpartum ovarian cyclic activity are not exactly clear, and there are some inconsistencies in the studies on this topic^13,26,27. Some studies found no link between serum glucose levels and the delay in postpartum ovarian cyclic activity^13,26,27. Others^28,29, however, argue that glucose is essential for normal ovarian dynamics in dairy cows, with cyclic cows showing much higher glucose levels than acyclic cows. The current study found no significant difference in glucose levels between the groups (p>0.05). These findings agree with those of Jeong et al.^13, Garverick et al.^26, and Obese et al.²⁷. The absence of changes in glucose concentrations is likely due to the strict homeostatic control over glucose levels³⁰.

Cholesterol is an important substrate for the synthesis of ovarian sex hormones and has a significant effect on follicular development. It has been reported that an imbalance in cholesterol homeostasis may have a negative impact on ovarian structure and function³¹. In the present study, although no significant difference was found in cholesterol and triglyceride levels (p>0.05), higher cholesterol levels were seen in cyclic cows. Cholesterol also plays a critical role in the synthesis of steroid hormones, which could indicate that hormonal activity is more intense in cyclic cows. This increase in cholesterol could be related to the support of follicular development and luteal function. It may also reflect the positive effects of effective nutritional interventions on lipid metabolism. Higher cholesterol levels may suggest higher hormonal activity, which is beneficial to reproductive health. Cholesterol is a precursor to the synthesis of steroid hormones such as progesterone and estrogen; therefore, higher levels could suggest increased hormonal activity³². Subtle differences in our findings suggest that lipid metabolism was regulated by successful nutritional interventions.

The resumption of ovarian cyclicity after calving is important for protecting reproductive health in dairy cows. It affects calving intervals and reproductive performance directly, which in turn affects the profitability of dairy farms^15,33. Delays in postpartum ovarian cyclic activity also extend the VWP, and cows with a longer VWP have lower calving frequency³⁴. Cows with regular postpartum ovarian cyclic activity are found to have better reproductive performance than cows with delayed postpartum ovarian activity¹⁴. Shrestha et al.¹⁴ reported that the calving to first insemination interval was 66.7 days in cyclic cows, compared to 92.7 days in anestrous cows. In the current study, the calving to first insemination interval was 72.55 days in cyclic cows and 172.7 days in acyclic cows. Both the first insemination and conception intervals were significantly earlier in cyclic cows. On the other hand, these intervals were delayed in acyclic cows. For cyclic cows, the calving to conception period was 87.36 days, and for acyclic cows, it was 187.70 days. Similar to our study, Ledoux et al.³⁵ and Gautam et al.³⁶ found that cows with delayed ovarian activity had longer calving to first insemination and calving to conception intervals than cows with normal ovarian function.

In conclusion, in the current study, no significant differences were found between cyclic and acyclic cows in terms of biochemical parameters, parity, or BCS. However, cyclic cows had significantly higher calving to first estrus, calving to first insemination, and calving to conception intervals compared to acyclic cows. These findings highlight the importance of monitoring cyclicity in herd management, further demonstrating that cows that return to cyclical activity at the end of the VWP have better reproductive performance.

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