​Color Doppler Ultrasonographic Characteristics of Embryonic Development and Mortality in Dairy Cows

Embryonic mortality in the reproductively sound animals, is one of the major factors of reproductive failure, decreased fertility, delayed pregnancy in cattle, fewer calves born, reduced milk production and slower genetic progress, especially, in repeat breeder females with late embryonic mortality (Diskin et al., 2016). Generally, fertilization rate with artificial insemination (AI) ranges from 85-90 per cent (Sreenan et al., 2001; Sharma et al., 2019), whereas, conception rate in dairy herds is about 52 per cent (Bansal et al., 2019) and declines an additional 10 per cent in lactating dairy cows (Whitlock and Maxwell 2008). Embryos are most susceptible to mortality during their development from morula to blastocyst stage i.e. the prehatching period (Shirasuna et al., 2012).
       
Color Doppler is a relatively new technique employed during recent times in cattle reproduction (Sharma et al., 2019a). Ultrasonography has been the best non-invasive method to study the characteristics of embryonic viability and mortality from day 25 post-AI (Sharma et al., 2018b) and also, the corpus luteum (CL) morphology (Ginther 2014) and functionality (Jokubkiene et al., 2006). Therefore, the present study envisaged the embryonic viability/mortality and luteal blood flow characteristics in dairy cows.

The study was conducted on twenty-nine (n=29) cross bred Jersey cows from March to December 2019 at Instructional Livestock Farm Complex (ILFC), CSK Himachal Pradesh Agricultural University, Palampur. Animals were housed in concrete dry sheds and fed with both green fodder and dry fodder/hay. Recommended dose of concentrate and mineral mixtures were supplemented in fodder/hay and clean drinking water was available free choice. All the animals had sound health with no apparent clinical signs of any disease or deficiency. Trans-rectal ultrasonography (Mindray Z5 VET) of the genital tract and ovaries was done with a linear rectal probe at 7.5 MHz frequency from day 21 to 42 of gestation at weekly intervals in brightness mode (B-mode) and color Doppler mode. Animals that were diagnosed as non-pregnant were scanned up to day 28 post insemination. Various characteristic echographic structures on ovaries and embryonic characteristics were visualized, recorded and analysed.
       
Corpus luteum (CL) was recorded on an ovarian map and its measurements were performed using ultrasound machine (internal digital callipers) for each examination. Blood perfusion to the corpus luteum was measured; cross-sectional images of the maximal area of the CL (square millimetre) as vascular transverse section were recorded. The mean CL vascularity was calculated as an average of three cross-sectional images using the grid technique (Luttgenau and Bollwein 2014).
       
The diagnosis of embryonic mortality was based on findings of Romano (2004) and Gabor et al., (2016). Numeric data for all the parameters are expressed as mean±SD and statistical analysis was carried out using one-way ANOVA (Statistical Analysis Software® 9.2 TS Level version 2M2 for windows).

In the present study, the overall incidence of embryonic mortality was recorded to be 25% with 15 and 10% mortality occurred between days 21-28 and 28-35 of gestation, respectively (Table 1). In accordance with our findings, Gatea et al., (2018) reported 23% embryonic mortality between days 30-60 of gestation, whereas, other researchers reported a lower incidence of 13% between days 32-45 (Fricke et al., 2016), 18.6-19.75% between days 24-42 (Pohler et al., 2016) and 17.0% between days 28-32 of gestation (Hernandez et al., 2012) in dairy cows. However, a higher incidence of 40-56% and 42.7% embryonic mortality in moderate and high yielding cows was reported between day 0-42 of gestation by Diskin and Morris (2008) and Pereira et al., (2013), respectively.
 

       
The size of CL was maximum at the beginning of examination (day 21), remained nearly constant over the period of examination in pregnant and embryonic mortality cows, but differed significantly (P<0.01) from non-pregnant cows (Table 2). In accordance with our findings, Luttgenau and Bollwein (2014) also reported a reduction in the CL size in cows that supervenes embryonic mortality during early gestation. Contrarily, Herzog et al., (2011) concluded that size of CL did not alter much after day 18 in cows with embryonic mortality i.e. after maternal recognition of pregnancy. The decrease in CL size is indicative of the loss of luteal parenchymal cells (Tamura et al., 2008) leading to an overall reduction in luteal tissue in subsequent examinations. Ultrasonographic studies of the corpus luteum revealed that it displays a dynamic pattern of development and regression based on the physiological state of the genitalia/state of pregnancy or non-pregnancy and embryonic mortality (Luttgenau and Bollwein 2014). Scully et al., (2014) observed a significant difference between CL size of pregnant and non-pregnant cows and considered it as a possible marker for a functional CL. However, some researchers do not consider the size of the CL as a reliable indicator of pregnancy and its functional status (Scully et al., 2014).
 

       
The vascularity of CL was initially along the outer border, in a ring-like pattern, which tends to increase towards the centre along with the increase in gestation length (Fig 1a, 2a, 3a, 4a). The vascularity in pregnant cows showed a higher percentage of luteal tissue with positive Doppler signals on initial examination (Day 21) differed significantly (P<0.01; P<0.05) from non-pregnant and embryonic mortality cows and continued to increase till the end of examination (Table 2). Also, the luteal tissue vascularity decreased constantly from day 28 until the end of examination in cows diagnosed with embryonic mortality (Fig 1b, 2b, 3b, 4b). In agreement with our findings, Scully et al., (2014) reported that pregnant cows have significantly higher vascularity as compared to non-pregnant cows on all days of examination. Similarly, Pinaffi et al., (2017) reported that vascularity of CL in pregnant cows tends to increase along gestation. The high vascularity demarcated the active functional state of CL for progesterone production (Luttgenau and Bollwein 2014). The CL blood flow is associated with luteal regression at end of the estrous cycle between day 16-20 post-ovulation (Pinaffi et al., 2017). Thus, the diagnosis of state of pregnancy as early as day 21 of gestation could be done on the basis of CL vascularity being present or absent (Pancarci et al., 2012).
 

 

 

 

       
The uterine horn lumen in pregnant cows increased throughout the period of examination (Table 2), along with the developing embryo proper with clear uterine fluid within the lumen (Fig 3a, 4a, 5a, 6a). The ultrasonographic appearance of uterine horns with uterine fluid was due to the activity of secretory glands and microvasculature associated with endometrial development and implantation of the embryo (Gray et al., 2001). The embryonic size had large variations among individual animals (Pohler et al., 2014). The embryo in pregnant cows was detected as early as 28 days of gestation (Fig  6a), with the size of the embryo increasing gradually in pregnant cows over the period of examination (Fig 7a, 8a). The amniotic sac, which surrounds the embryo, increased concurrently with the size of the embryo in pregnant cows (Table 2). Therefore, diagnosis of pregnancy based on ultrasonographic visualization of the embryo was possible only 28 days after successful AI, as the embryo lies closely adhered to the uterine wall which is concurrent with our findings (Gnemmi 2004).
 

 

 

 

 
The cows diagnosed with embryonic mortality showed distinct ultrasonographic signs from day 28 of gestation, which included reduced intra-uterine allantoic fluid and absence of embryo proper with an embryonic heartbeat (Fig 8b) in contrast to the findings recorded in pregnant cows (Fig 8a). Uterine lumen was filled with hyperechoic free-floating structures representing embryonic debris (Fig 6b, 7b, 8b), embryonic remnants with ill-defined embryonic outline and distorted allanto-chorionic membrane (Fig 6b, 7b, 8b). The intra-uterine fluid, echogenic free-floating structure and embryonic debris within the uterine lumen reduced over the period of examination (Fig 6b, 7b, 8b) and got reabsorbed within varying time indifferent cows. The cows with embryonic mortality show various ultrasonographic signs which include reduced intra-uterine allantoic fluid (Drews et al., 2012) and absence of embryo proper with an embryonic heartbeat (Flores et al., 2014). The lumen was filled with hyperechoic free-floating structures, representing embryonic debris (Drews et al., 2012; Flores et al., 2014), embryonic remnants with an ill-defined embryonic outline and distorted allanto-chorionic membrane (Lopez-Gatius and Garcia-Ispierto, 2010) which are in unison with our findings. The intra-uterine fluids reduced over time (Flores et al., 2014) and got reabsorbed by the maternal immune system.
 
As a part of peroration, color Doppler ultrasonography has been very effective in the differential diagnosis of physiological (pregnant/non-pregnant) conditions as well as embryonic mortality based on CL size, CL vascularity, and embryonic characteristics.

Authors would like to thank head of Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, CSKHPKV, Palampur for providing all the necessary facilities during the present study.

Authors declare no conflict of interest.