Ovarian activity and embryo yield in relation to the postpartum period in superovulated dairy cows

The aim of this study was to evaluate superovulation response in cows at various postpartum periods (early postpartum period up to 3.5 months; middle postpartum period 3.7–7 months; later postpartum period above 7.5 months after calving). The data included observation of 55 Holstein cows superovulated at one farm in the Czech Republic during the years 2010 and 2013. Reproduction traits (dependent variable) were represented as number of the corpora lutea, number of transferable embryos, morulae, blastocysts, total number of embryos and embryo recovery. For statistical evaluation we used the PROC GLM of SAS® with fixed effect breeding value of milk production. The study results show significant differences (P < 0.05–0.01) in the three postpartum periods (early, middle, and later postpartum periods) and the number of corpora lutea (4.6; 7.4; 10.8), number of total embryos (3.2; 2.9; 6.5) and transferable embryos (1.8; 1.7; 4.4). Effective timing of embryo transfer in the later postpartum period resulted in greater ovarian activity and embryo yield compared to early lactation periods. Superovulation, corpus luteum, transferable embryos Milk production and reproduction are two important physiological traits that have consistently demonstrated a negative correlation in cows (Stádník and Louda 1999) due to the physiological adaptation of cows to excessive milk production in recent years (Lucy 2001). Both traits are related to individual genetics (Bezdíček et al. 2007). However, deteriorating reproduction in recent years is also associated with the level of inbreeding (Bezdíček et al. 2008; Bezdíček et al. 2014) and the quality of the environment has an important impact, too. Good reproduction in dairy cows is associated with a period of sharp increase in milk production in early lactation, connected to increased demands on the metabolism of the whole organism (Stádník et al. 2015) in maintaining the physiological condition of the cow (Stádník et al. 2002) and allowing utilization of energy resources to provide the necessary endocrine indicators (Royal et al. 2000; Beran et al. 2013). The prenatal period, birth, and the start of the lactation period are significantly associated with large energy demands on the body. The results of a number of studies have shown that in early lactation, animals are under greater physiological stress than in later postpartal periods. Bauman and Currie (1980) reported that cows use aproximately 50 kg of pure lipid of body energy reserves during the first 10 weeks of lactation. Villa-Godoy et al. (1988) pointed out the fact that the caloric deficit can be one reason for infertility in lactating ACTA VET. BRNO 2017, 86: 51–57; https://doi.org/10.2754/avb201786010051 Address for correspondence: Luděk Stádník Czech University of Life Sciences Prague Kamýcká 129, 165 21, Praha 6 – Suchdol, Czech Republic Phone: +420 224 383 057 E-mail: stadnik@af.czu.cz http://actavet.vfu.cz/ dairy cows. Butler (2000) added that a negative energy balance can delay the time of first ovulation through inhibition of the luteal hormone (LH) and low concentrations of blood glucose, insulin, and insulin-like growth factor-I (IGF-I) followed by inhibition of oestrogen production by dominant follicles. Loker et al. (2009) found that cows with ≤ 180 days open tended to have low yields in the last part of lactation. Cows with longer days open had a proportionally higher milk yield throughout lactation, suggesting a possible confounding effect of prolonged days open. Maillo et al. (2012) studied the direct effect of lactation (lactating and dry cows) on embryo development in Holstein cows, reporting that 26.3% of total embryos developed to the blastocyst stage in the lactating cows compared to 39.6% in the nonlactating groups. Leroy et al. (2005) who compared embryo quality in lactating Holstein cows, non-lactating Holstein heifers, and Belgian Blue beef cows concluded that lactating cows yielded embryos with a significantly reduced quality (only 13.1% embryos categorized as excellent vs. 62.5% in non-lactating heifers, and 55.0% in Belgian Blue cows). A lower percentage of blastocyst stage (4%) was also found in these dairy cows compared to 23.2% in heifers and 17.3% in Belgian Blues. Sartori et al. (2002) found lower embryo quality in lactating cows compared to dry cows (3.1 vs 2.2 points; on a scale of 1 = excellent to 5 = degenerate), as well as in lactating cows compared to heifers (3.8 vs 2.2 points). Rizos et al. (2010) also reported that the reproductive tract of a postpartum lactating dairy cow may be compromised in supporting early embryo development compared to nonlactating heifers. This conclusion was based on a higher proportion of embryos recovered from heifers (79.0 vs 57.2%) as well as on lower progesterone concentrations in lactating cows (d 7; 2.39 vs 5.34 ng/ml). Walters et al. (2002) found that dairy cows produced a higher proportion of good (+) oocytes at day 119 than cows at day 32 postpartum. Aspiration of oocytes took place in these two groups (early lactation, mid-lactation) and in the following days (twice weekly, over a 10-week period). The results showed a linear increase in the number of follicles in both groups. The number of follicles increased from 14.2 (Day 119) to 18.1 (Day 190) in mid-lactation and from 14.9 (Day 32) to 15.7 (Day 90) in early-lactation. This fact indicates that the length of postpartum could have an important effect. Most research has been focused on standard reproduction characteristics, e.g., the service period, number and quality of embryos or oocytes. Evaluation of the corpus luteum count as an important informative trait is still missing. For these reasons, the objective of this study was to evaluate the effect of the postpartum period of different lengths (early postpartum period up to 3.5 months; middle postpartum period of 3.7–7 months; later postpartum period above 7.5 months) on the superovulation response in Holstein dairy cows determined by the number of corpora lutea and the number and quality of flushed embryos. Materials and Methods The sample included 55 Holstein cows (aged 1.4 to 7.8 years) superovulated at one farm in the Czech Republic during the years 2010 and 2013. The farm was located in the lowlands. Average milk production (in the 1st lactation) at the time of the experiment was as follows: 7 982 kg of milk; 4.01% of fat; 320 kg of fat; 3.30% of protein; 264 kg of protein . Oestrus was synchronized by injection of a prostaglandine F2α analogue – oestrophan (Bioveta a.s., Ivanovice na Hane, Czech Republic). The cows were superovulated by injection of porcine pituitary gonadotropin (Pluset ®FSHp-LHp, Laboratorios Callier, Barcelona, Spain) twice daily for 5 days at 08:00 and 20:00 h given in decreasing doses starting with doses of 150 IU follicle stimulating hormone (FSH) +150 IU luteal hormone (LH) in the morning on day 11 to 50 IU FSH+50 IU LH in the evening on day 15 of the oestrous cycle. On day 13, oestrophan was administered for luteolysis. Insemination was performed × 4 with frozen-thawed semen at 52

Milk production and reproduction are two important physiological traits that have consistently demonstrated a negative correlation in cows (Stádník and Louda 1999) due to the physiological adaptation of cows to excessive milk production in recent years (Lucy 2001).Both traits are related to individual genetics (Bezdíček et al. 2007).However, deteriorating reproduction in recent years is also associated with the level of inbreeding (Bezdíček et al. 2008;Bezdíček et al. 2014) and the quality of the environment has an important impact, too.Good reproduction in dairy cows is associated with a period of sharp increase in milk production in early lactation, connected to increased demands on the metabolism of the whole organism (Stádník et al. 2015) in maintaining the physiological condition of the cow (Stádník et al. 2002) and allowing utilization of energy resources to provide the necessary endocrine indicators (Royal et al. 2000;Beran et al. 2013).
The prenatal period, birth, and the start of the lactation period are significantly associated with large energy demands on the body.The results of a number of studies have shown that in early lactation, animals are under greater physiological stress than in later postpartal periods.Bauman and Currie (1980) reported that cows use aproximately 50 kg of pure lipid of body energy reserves during the first 10 weeks of lactation.Villa-Godoy et al. (1988) pointed out the fact that the caloric deficit can be one reason for infertility in lactating dairy cows.Butler (2000) added that a negative energy balance can delay the time of first ovulation through inhibition of the luteal hormone (LH) and low concentrations of blood glucose, insulin, and insulin-like growth factor-I (IGF-I) followed by inhibition of oestrogen production by dominant follicles.Loker et al. (2009) found that cows with ≤ 180 days open tended to have low yields in the last part of lactation.Cows with longer days open had a proportionally higher milk yield throughout lactation, suggesting a possible confounding effect of prolonged days open.Maillo et al. (2012) studied the direct effect of lactation (lactating and dry cows) on embryo development in Holstein cows, reporting that 26.3% of total embryos developed to the blastocyst stage in the lactating cows compared to 39.6% in the nonlactating groups.Leroy et al. (2005) who compared embryo quality in lactating Holstein cows, non-lactating Holstein heifers, and Belgian Blue beef cows concluded that lactating cows yielded embryos with a significantly reduced quality (only 13.1% embryos categorized as excellent vs. 62.5% in non-lactating heifers, and 55.0% in Belgian Blue cows).A lower percentage of blastocyst stage (4%) was also found in these dairy cows compared to 23.2% in heifers and 17.3% in Belgian Blues.Sartori et al. (2002) found lower embryo quality in lactating cows compared to dry cows (3.1 vs 2.2 points; on a scale of 1 = excellent to 5 = degenerate), as well as in lactating cows compared to heifers (3.8 vs 2.2 points).Rizos et al. (2010) also reported that the reproductive tract of a postpartum lactating dairy cow may be compromised in supporting early embryo development compared to nonlactating heifers.This conclusion was based on a higher proportion of embryos recovered from heifers (79.0 vs 57.2%) as well as on lower progesterone concentrations in lactating cows (d 7; 2.39 vs 5.34 ng/ml).Walters et al. (2002) found that dairy cows produced a higher proportion of good (+) oocytes at day 119 than cows at day 32 postpartum.
Aspiration of oocytes took place in these two groups (early lactation, mid-lactation) and in the following days (twice weekly, over a 10-week period).The results showed a linear increase in the number of follicles in both groups.The number of follicles increased from 14.2 (Day 119) to 18.1 (Day 190) in mid-lactation and from 14.9 (Day 32) to 15.7 (Day 90) in early-lactation.This fact indicates that the length of postpartum could have an important effect.Most research has been focused on standard reproduction characteristics, e.g., the service period, number and quality of embryos or oocytes.Evaluation of the corpus luteum count as an important informative trait is still missing.
For these reasons, the objective of this study was to evaluate the effect of the postpartum period of different lengths (early postpartum period up to 3.5 months; middle postpartum period of 3.7-7 months; later postpartum period above 7.5 months) on the superovulation response in Holstein dairy cows determined by the number of corpora lutea and the number and quality of flushed embryos.

Materials and Methods
The sample included 55 Holstein cows (aged 1.4 to 7.8 years) superovulated at one farm in the Czech Republic during the years 2010 and 2013.The farm was located in the lowlands.Average milk production (in the 1 st lactation) at the time of the experiment was as follows: 7 982 kg of milk; 4.01% of fat; 320 kg of fat; 3.30% of protein; 264 kg of protein .
Oestrus was synchronized by injection of a prostaglandine F2α analogue -oestrophan (Bioveta a.s., Ivanovice na Hane, Czech Republic).The cows were superovulated by injection of porcine pituitary gonadotropin (Pluset ® -FSHp-LHp, Laboratorios Callier, Barcelona, Spain) twice daily for 5 days at 08:00 and 20:00 h given in decreasing doses starting with doses of 150 IU follicle stimulating hormone (FSH) +150 IU luteal hormone (LH) in the morning on day 11 to 50 IU FSH+50 IU LH in the evening on day 15 of the oestrous cycle.On day 13, oestrophan was administered for luteolysis.Insemination was performed × 4 with frozen-thawed semen at 12 h intervals started at 12 h after detection of the standing oestrous.Doses of semen from one sire produced in one batch were used.Embryo recovery was performed on day 7 after the first insemination by a standard non-surgical technique to flush out the uterine horns.Uterine flushing was conducted with a complete flush solution (Bioniche, Belleville, Ontario, Canada) using a silicone two-way Foley catheter (Minitüb GmbH, Tiefenbach, Germany).Flushed ova and embryos were transferred to the holding medium -phosphate buffered solution (PBS) with 20% foetal calf serum (FCS, Gibco BRL, USA) and assessed using a stereomicroscope.The embryos were evaluated according to their stage of development as transferable (i.e., morulas, blastocysts) or non-transferable (degenerated and unfertilized).
To determine the number of corpora lutea (CL), both ovaries were examined with sonograph Tringa Linear (Canmedical, Canada).
To evaluate the results of superovulation, the animals were divided at the start of synchronization and subsequent superovulation into three groups according to the length of the postpartum season (EPP -early postpartum period to 3.5 months; MPP -middle post-partum period 3.7-7 months; LPP -later postpartum period above 7.5 months after calving).The experimental cows were divided into groups according to the postpartum periods.
The PROC GLM of SAS ® with fixed effect (breeding value of milk production) was applied to all data.Breeding values of milk production were based on the actual results from January 2015.The use of breeding values was convenient because the breeding value is the key indicator reflecting the individuals in production.Breeding values varied from -253 to +1 155.
The effects of the postpartum period were estimated from the model as follows: y ij = μ + a i + b×(BVM) + e ij where: y ij -corrected value (dependent variable) represented the number of corpora lutea, transferable embryos, morulae, blastocysts, total number of embryos and embryo recovery, μ -mean value a i -fixed effect of postpartum period (i = 1, n = 19; i = 2, n = 20; i = 3, n = 15); b×(BVM) -regression on the breeding value for milk production e ij -residual error Differences between the estimated variables were tested at the level of significance P < 0.05 (a; b) or P < 0.01 (A; B).

Results
The results (Table 1) show significant differences (P < 0.05-0.01)between the three postpartum period groups (EPP -early postpartum period to 3.5 months; MPP -middle postpartum period of 3.7-7 months; LPP -later postpartum period above 7.5 months after calving) and the superovulation response -represented by the number of corpora lutea (4.6; 7.4; 10.8) calculated as the regression analysis with fixed effect of the breeding value for milk production.
The trend (Fig. 1) to higher counts of corpora lutea after superovulation treatment in the later postpartal period (LPP) than in early lactation (EPP) also corresponds with the total count of embryos (3.2; 2.9; 6.5) and with the morula counts too (1.2;1.8; 3.5).The results also showed significantly (P < 0.05) higher counts of embryos (better results) in the later (LPP) than in the early (EPP or MPP) postpartum period.
In terms of quality of the embryos (transferable embryos), significantly better results were found in the later postpartal period than in the middle or early postpartum period (1.8; 1.7; 4.4; P < 0.05).On the other hand, we found no significant differences between blastocyst counts (1.9; 0.6; 1.5) or embryo recovery (56.8; 34.3; 53.7%).
We also calculated the coefficient of determination (representing the share of explained variance), which was the greatest number of corpora lutea (r 2 = 0.41).For the other traits there was a coefficient of determination between r 2 = 0.20-0.29 (Table 2).

Discussion
Generally, the results show that the superovulation response (represented by the number of corpora lutea) was significantly greater during the time period with lower loading of the organism in terms of milk production (LPP).There was a significantly lower response of the organism to superovulation during the first phase of lactation (EPP) than in the period with less energy-loading.These findings generally agree with Makarevich et al. (2016).
Similar findings were reported by Walters et al. (2002).These authors also showed increased number of follicles from 14.2 (Day 119) to 18.1 (Day 190) in middle-lactation, whereas in early-lactation the number of follicles increased from 14.9 (Day 32) to 15.7 (Day 90).In agreement with our study, the authors reported a higher percentage of good (+) oocytes at day 119 (4.1 ± 0.9%) than in cows at day 32 postpartum (1.6 ± 0.9%) (Walters et al. 2002).
In agreement with our findings, Kendrick et al. (1999) presented that oocyte number increased (linearly) from days 30 to 100 postpartum.At the same time the authors found that the cows produced more good oocytes when they were fed high energy diets.Lucy et al. (1991) also found a significant relation between energy balance and follicular and ovarian function in the postpartum period in Holstein cows.The authors report that the number of large follicles (from 10 to 15 mm) detected by ultrasonography increased when the energy balance of cows increased.At the same time with the increasing energy balance, the number of small-sized follicles (from 6 to 9 mm) in Holstein cows decreased (Lucy et al. 1991).Snijders et al. (2000) studied the effect of genetic merit (milk yield, body condition) on in vitro oocyte development in dairy cows.The authors found that fewer blastocysts and lower cleavage was conected with a high genetic merit of cows.On the other hand, Matoba et al. (2012) found a positive association between the milk yield and bodyweight with regard to the recovered number of oocytes.The authors did not find an effect of the lactation metabolic stress on oocytes.
De Bie et al. ( 2016) also hypothesized that negative energy balance has a negative effect on oocyte quality in Holstein Friesian cows.The authors report that the addition of β-carotene can improve β-carotene availability in the oocytes and hereby the oocyte quality in dairy cows.Beran et al. (2012) reported a significant effect of the sire on fertilization ability as well as pregnancy rate of cows.
Milk production is generally associated with high energy demands on animals.This is reflected in reproduction, as reported by a number of authors who describe poorer results in high production in contrast to low-lactating cows.Sartori et al. (2002) found lower embryo quality in lactating cows compared to dry cows (3.1 vs 2.2) and also comparing lactating cows and non-lactating heifers (3.8 vs 2.2).Rizos et al. (2010) also report more embryos recovered from non-lactating heifers than from lactating cows (79.0 vs 57.2%).A lower proportion of embryos reaching the blastocyst stage in all lactating cows (4%) compared to 23.2% in non-lactating heifers was also described by Leroy et al. (2005).EPP -early postpartum period (up to 3.5 months); MPP -middle postpartum period (3.7-7 months); LPP -later postpartum period (above 7.5 months) The same superscript within the column means significant differences -P < 0.05 (a; b) or P < 0.01 (A; B) They determined significantly reduced embryo quality in lactating cows compared to nonlactating ones as well.
Early lactation is a period characterised by an intensive increase in milk production and high energy demands on the organism.For this reason, this period is associated with negative energy balance (Roche et al. 2007), fat mobilisation and a decrease in the body condition score (BCS).In this way, the level of metabolism significantly influences reproduction as an energy deficit which can lead to infertility (Villa-Godoy et al. 1988).
The aim of the present study was to evaluate the reproduction efficiency of cows in various postpartal periods with significantly different metabolic loads.The results demonstrate that the effects of the postpartal period on reproduction are very important; e.g., in terms of effective timing of embryo transfer, when in periods with a lower metabolic load of the organism (later postpartum period), higher ovarian activity and embryo yield can be expected than in the early lactation periods.The energy balance of the organism is also very important for the right timing of insemination.In conclusion, significant differences in ovarian activities after superovulation represented by numbers of corpora lutea in relation to the length of the postpartum period were determined.The results show that the superovulation response represented by the number of corpora lutea as well as by the number and quality of embryos was significantly higher during a lower load of the organism in terms of milk production.
Influence of length of the postpartal period on ovarian activity, yield and quality of the flushing embryos.

Fig. 1 .
Fig. 1.Relationship of different postpartum period lengths and reproduction in dairy cows.

Table 2 .
The coefficients of determination and significance of various factors included in the model.