Embryo Transfer as an Option to Improve Fertility in Repeat Breeder Dairy Cows.

Repeat breeding is a serious reproductive disorder in dairy cattle. The causes of repeat breeding are multifactorial and there are two main mechanisms: failure of fertilisation or early embryo death, mainly due to poor quality of oocytes and an inadequate uterine environment. Many methods have been used to increase the pregnancy rate for repeat breeder cows, such as intrauterine infusion of antibacterial agents or antibiotics, hormonal treatments for oestrus synchronisation and induction of ovulation, and progesterone supplementation or induction of accessory corpus luteum; however, the results were inconsistent between studies. Embryo transfer (ET) has the capability to minimalise the effects of poor oocyte quality and unfavourable uterine environments on early embryo development during the first seven days after ovulation in repeat breeder cows, and several studies showed that ET significantly improved the pregnancy rate in this group of animals. Thus, ET can be considered an option to increase the conception rate in repeat breeder dairy cows.

Definition and occurrence of repeat breeding

Repeat breeding is one of the major problems in dairy cows. Repeat breeders are cows without any anatomical or infectious abnormalities that do not become pregnant after three or more breeding attempts or many artificial inseminations (AI) (16, 25). The incidence of repeat breeding in cattle has been reported as 9.0% in the UK (9), 24.0% in the USA (6), 10.1% in Sweden (25), 12.4% in Poland (31), and 25.1% in Spain (21). Repeat breeding decreases dairy profit because of wasted semen and insemination expenditure, longer inter-calving periods and higher veterinary treatment, culling and replacement costs (6, 11, 38, 48).

The causes of repeat breeding

The causes of repeat breeding are multifactorial. The failure of fertilisation or early embryo death are its two main mechanisms (13, 49) (Fig. 1); however, fertilisation does not seem to be the principal factor responsible for repeat breeding. Sartori et al. (55) reported that the fertilisation rates are 83% in cows and more than 90% in heifers, but these are not equalled by pregnancy rates. Fertilisation can fail because of poor oocyte quality or problems associated with AI, one of which is insemination at an inappropriate time. Fertilisation failure can also be caused by chromosomal abnormalities, heat stress and endocrine problems induced by high milk production or unbalanced nutrition (55, 68). There are many studies confirming that oocytes in repeat breeder cows are poorer in quality than those in healthy cows and that it is this oocyte quality deficit that obstructs fertilisation or causes embryo mortality (19, 32, 37, 62). An example is the research by Gustafsson and Larsson (26), in which 74% of the embryos from superovulated virgin heifers on day 7 after insemination were morphologically normal compared with only 28% from superovulated repeat breeder heifers. Oocytes originating from high-yielding cows during lactation were found to develop into embryos of poorer quality than oocytes from heifers, non-lactating cows, or cows with medium milk yields (39, 55, 60). Other researchers (3, 6) found the hormonal asynchrony suprabasal progesterone levels and delayed LH peaks around oestrus in repeat breeder cows, and these deviations prolonged the lifespan of preovulatory follicles and negatively affected the final maturation of oocytes (3, 6). Fertilisation failure can be also caused by inadequate oestrus detection leading to improper time of AI, incorrect AI technique, low semen quality and obstructed oviducts (55, 68, 73).

Fig. 1

Possible causes of repeat breeding

Embryo death is the major cause of reproductive failure. Estimated ruminant embryonic mortality ranges between 20 and 50% (14, 30). In high-producing dairy cows, the highest embryo loss occurs within the first week post conception (15, 56). The causes of early embryo mortality to day 7 post conception centre on the early embryo’s inability to develop as a consequence of poor oocyte quality or an inadequate uterine environment associated with nutritional factors, heat stress, endocrine imbalance and uterine diseases (35, 63, 68). An excess of dietary protein has a detrimental effect on the uterine environment as it raises the level of ammonia in the blood and uteri of cows (10). Heat stress has deleterious effects on oocyte maturation and the developmental competence of preimplantation embryos due to elevated maternal body temperature (55, 59, 70). A low progesterone concentration due to its increased metabolism in high-yielding cows impoverishes the uterine environment such that it is unable to support early embryonic development (40, 69). Endometritis may induce embryo death by endometrial damage, bacterial toxins and inflammatory mediators such as prostaglandins, nitric oxide, reactive oxygen species and cytokines (58, 67); a high prevalence of subclinical endometritis (SE) has been reported in repeat breeder cows (31, 50, 54).

Treatment of repeat breeder cows

Numerous treatments have been studied to improve fertility in repeat breeder cows. Since repeat breeding may be related to subclinical endometritis, intrauterine infusions of antibiotics are commonly used for the treatment of cows in this group. However, some results were controversial (1, 20, 47). A few studies on the treatment of repeat breeders with an intrauterine infusion of antibacterial agents or antibiotics 24 h after insemination resulted in varying degrees of success (23, 24, 57). Treatment with nonsteroidal anti-inflammatory drugs at the time of AI did not influence the conception rate (27). The use of PGF2α for the treatment of subclinical endometritis was also tested; however, the effects were variable (33, 41).

There have been many studies on hormonal treatment for ovulation disorders in repeat breeders. Stevenson et al. (64) compared the effects of double insemination during the same oestrous period and injection of GnRH at the time of AI on pregnancy rates of repeat breeder dairy cows. Gonadotropin-releasing hormone significantly increased the pregnancy rates of repeat breeders (41.6 vs 32.1%), while double AI failed to improve them. Morgan and Lean (45) performed a meta-analysis of 40 trials on the administration of GnRH at AI and reported that the pregnancy rate for repeat breeder cows increased by 22.5%. Mee et al. (43) proposed a GnRH injection for repeat breeder cows 12 hours after the onset of oestrus. The number of pregnant cows having received GnRH was higher than that of control cows administered only saline (43 vs 14.%). Some studies revealed that the conception rate was improved in repeat breeder cows treated with Ovsynch protocols (2, 36).

Progesterone insufficiency in the early luteal phase is associated with embryo mortality in cows. Many methods have been tried to increase the conception rate by enhancing the endogenous progesterone level in repeat breeder cows. A number of studies have investigated the effects of progesterone supplementation on pregnancy rates. A meta-analysis by Yan et al. (72) gave very variable results. Progesterone supplementation between days 3 and 7 post insemination was beneficial only in lower fertility cows treated at spontaneous oestrus. Progesterone concentration can also be increased through induction of an accessory corpus luteum by human chorionic gonadotropin (hCG) treatment on day 5 after AI or GnRH treatment 11–14 days after it. However, the results were not consistent between studies (46, 49). Another method for supplementing progesterone after AI is the use of a progesterone-releasing intravaginal device (PRID). Villarroel et al. (66) assessed the efficacy of supplementation with exogenous progesterone for 14 days on pregnancy maintenance in inseminated repeat breeder cows but they found no positive effect of the PRIDs which they used on the pregnancy rate.

ET in repeat breeder cows

ET is widely used to increase the number of high-yielding animals and create genetically superior stock. Many studies have also investigated the use of ET for improving fertility in repeat breeder cows (Table 1).

Table 1

Conception rate in repeat breeder cows after AI or ET

Author	Conception rate		Comments
	AI	ET
Tanabe et al. 1985 (65)	-	70%	fresh embryos transferred surgically
Rodrigues et al. 2007 (52)	17.9%	41.7%	frozen-thawed embryos
Son et al. 2007 (61)	heat 7.7% vs TAI 18.5%	53.8%	frozen-thawed embryos; TET with CIDR and EB
Dochi et al. 2008 (16)	20.4%	41.5%	frozen-thawed IVF embryos following AI
Block et al. 2010 (7)	TAI 31.3%	50.5% vs 27.7%	fresh embryos vs vitrified embryos (in vitro); TET
Rodrigues et al. 2010 (53)	-	42.9% vs 38.2%	frozen-thawed embryos; TET with norgestomet implant and eCG (cows with or without CL on day 0 of the protocol)
Canu et al. 2010 (11)	30.0%	52.6%	frozen-thawed embryos (92%) and fresh embryos (8%) following AI after natural heat
Yaginuma et al. 2019 (71)	-	46.9%	frozen-thawed IVF embryos following AI

AI – artificial insemination; CIDR – controlled internal drug release; CL – corpus luteum; EB – estradiol benzoate; eCG – equine chorionic gonadotropin; ET – embryo transfer; IVF – in vitro fertilisation; TAI – timed artificial insemination; TET – timed embryo transfer

Tanabe et al. (65) compared the fertility of normal and repeat breeder cows as embryo recipients. Fresh embryos were transferred surgically into uteri, and groups were monitored for gravidity. There were no significant differences in pregnancy rates on day 60 between normal and repeat breeder recipients (82% and 70%, respectively). Rodrigues et al. (52) compared pregnancy rates in 5.693 repeat breeder Holstein cows after AI and 3.858 cows after ET. Pregnancy rates were greater after ET (41.7%) than after AI (17.9%).

To avoid the need for oestrus detection during ET procedure, an alternative is timed AI (TAI) in superovulated donors and timed embryo transfer (TET) in embryo recipients. Son et al. (61) evaluated pregnancy rates following controlled internal drug release (CIDR) TAI or TET protocols compared with the rates following AI after a single PGF2α injection in the luteal phase (8–13 days after oestrus) and AI after oestrus in lactating repeat breeder dairy cows. Cows at random stages of the oestrous cycle received the CIDR device and 2 mg estradiol benzoate (EB) (day 0), a PGF2α injection at the time of CIDR removal on day 7 and a 1 mg EB injection on day 8 for ovulation synchronisation. The cows then received TAI on day 9, 30 h after the second EB injection or TET on day 16 using frozen-thawed embryos. The pregnancy rate was significantly higher in the ET group (53.8%) than in the group for AI at detected oestrus (18.5%) or the TAI (7.7%) group. However, EB is banned in EU countries. Block et al. (7) investigated the proportion of cows pregnant following timed transfer of either fresh or vitrified embryos produced in vitro compared with the same proportion following timed AI. The pregnancy rate was 31.3% for cows subjected to TAI, 50.5% for cows receiving fresh embryos, and 27.7% for cows receiving vitrified embryos. Embryo transfer was particularly efficacious for infertile cows that had previously experienced several failed breeding attempts. The cows bred &3 times had a significantly lower rate of pregnancy success than cows bred ≤3 times if the cows were inseminated, but not if the cows received a fresh or vitrified embryo. Rodrigues et al. (53) proposed a new protocol for fixed-time embryo transfer in repeat breeder cows with the addition of equine chorionic gonadotropin to the ovulation synchronisation using a norgestomet implant. Only cows with confirmed corpus luteum (CL) prior to transfer were selected as recipients. The conception rates for cows with and without CL on day 0 of the protocol were 42.9% and 38.2%, respectively.

Some studies showed that embryo transfer following AI increased pregnancy rates in repeat breeder cows compared with AI alone. It is speculated that the higher pregnancy rate of ET following AI in repeat breeder cattle is due to the increased release of interferon tau (IFNT) from the added embryos. A greater amount of IFNT could support maternal recognition of pregnancy. Dochi et al. (16) investigated the success of AI alone or in combination with transfer of embryos produced in vivo to overcome repeat breeding problems. In repeat breeder cows, the pregnancy rate for AI alone was 30.0% compared with 52.6% after AI with embryo transfer. In the study of Yaginuma et al. (71), 1,122 repeat breeders were implanted with IVF embryos after previous AI. Implantation following insemination resulted in a pregnancy rate of 46.9% in repeat breeders. Added embryos increased the mRNA expression of interferon stimulated genes, indicating this pathway as the main mechanism leading to maintenance of embryos and resulting in a higher pregnancy rate achieved with such a protocol. These studies showed a relatively high (6.25 - 18.4 %) twin occurrence after ET following AI, while the twinning rate in dairy cows after AI alone was 0.3 - 5.0% (22, 44). Twin calving has several negative consequences for both cows and calves, such as increased incidence of abortion, dystocia, higher mortality of calves around or during parturition, increased incidence of placenta retention, metabolic disorders, and decrease in milk production (22).

Apparently, ET has the potential to overcome poor oocyte and embryo quality and the deleterious effects of uterine inadequacy on early embryo development during the first seven days in repeat breeder cows. Both oocyte and embryo quality and uterine environment are adversely affected by many factors such as negative energy balance, body condition loss and metabolic diseases (40, 51, 60), heat stress (55, 70) and subclinical endometritis (SE) (58, 67). This type of endometritis is characterised by inflammation in the absence of clinical signs and is defined by polymorphonuclear neutrophil (PMN) content exceeding 5% in samples collected by endometrial cytobrush (42). Prevalence of SE in repeat breeder cows ranging from 12% to 53% has been reported. Several studies showed negative effects of SE on fertility after AI (4, 5, 8, 34, 54), and the same have been described on embryo quality in donor cows. Carvalho et al. (12) and Fernandez-Sanchez et al. (18) reported a significant reduction in the percentage of viable embryos in cows with high endometrial PMN at the onset of superovulation. Drillich et al. (17) investigated PMN dynamics in the endometrium of donor cows from AI to the time of flush (day 7) and found a significant relationship between them and the flushing outcome. Cows without PMNs at AI but with them at flush yielded the highest number of transferable embryos. Hoelker et al. (29) reported that SE affected gene expression in embryos, including the expression of genes related to membrane stability, the cell cycle and apoptosis.

However, there are no studies on the effect of SE on pregnancy outcomes after ET. It is assumed that inflammation causes an abnormal uterine environment and disrupts embryo survival (58). Hill and Gilbert (28) showed a reduction in the quality of embryos cultured in media conditioned by fluid from inflamed uteri. In the studies presented in this review (2, 3, 16, 19, 37, 39, 52, 53, 61, 62, 64, 65, 66, 71), repeat breeder cows were not differentiated regarding the occurrence of SE. It is likely that pregnancy rates would be higher in cows without SE. Endometrial cytology is considered the most reliable method for the diagnosis of SE (5, 42) and can be used to select recipient cows without the condition. The technique is appropriate for use in the additional studies which are needed to evaluate the correlation between the effectiveness of ET in repeat breeder cows and endometrial PMN count.

The results of the presented studies are encouraging and indicate that ET improves the pregnancy rate in repeat breeder cows by minimising the impact of poor oocyte quality and inadequate uterine environments on fertilisation and embryo development during the first 7 days after AI. Thus, ET can be considered an option to improve fertility in repeat breeder dairy cows. However, it should be noted that ET cannot be more than a course of action to improve the pregnancy rate; parturition and feeding should be still carefully organised in a way best suited to that group of animals and their uterine health requires vigilant management.