Hydrops in a heifer as a result of in-vitro fertilization

Abstract

A 2-year-old Holstein heifer was presented at 7 months gestation with a 2-day history of premature mammary enlargement. It was suspected that the dam was showing clinical symptoms of a pending abortion. Through diagnostic testing and postmortem examination, a final diagnosis of hydrops allantois and amnion was determined.

A 2-year-old Holstein heifer was presented to the Atlantic Veterinary College (AVC) at 224 days gestation with a 2-day history of premature mammary enlargement. The owner was concerned that the heifer may be aborting. The heifer was the recipient of an in-vitro produced embryo.

Case description

On presentation, the 592-kg heifer was calm, alert, and responsive. Apart from a slightly elevated heart rate (100 beats/min; normal range: 60 to 80 beats/min) and respiratory rate (37 breaths/min; normal range: 10 to 30 breaths/min) parameters were within normal limits. There was moderate, symmetrical mammary gland development with pitting edema overlying the gland and ventral abdomen. The abdominal girth of the dam appeared to be larger than expected for this stage of gestation.

On rectal examination, a large fluid-filled uterus could be palpated that was displaced to the right of midline. Transrectal ultrasonography confirmed the rectal examination findings and identified a thick amnion containing echogenic fluid. There were fewer placentomes than expected and their size was smaller than expected. The conceptus was not visualized during transrectal ultrasonic imaging; thus, transabdominal ultrasonography was performed. On transabdominal evaluation, the conceptus was alive and active. The fetal heart rate was 115 beats/min (normal range: 106 to 130 beats/min). Moderate ascites was evident in the fetus allowing visualization of a highly echogenic liver. The fetal membranes were markedly thickened and contained echogenic fluid.

Although a serum sample revealed a normal progesterone concentration (2.1 ng/mL), a progesterone-releasing intravaginal device (PRID) (Vétoquinol, Lavaltrie, Quebec), 1.55 g was inserted to help prevent abortion. Analysis of a free catch urine sample revealed a normal specific gravity (1.013) with traces of blood and protein.

Over the next 24 h, the heifer was monitored at least twice daily to assess body weight and girth. The fetus was examined using transabdominal ultrasonography for movement and heart rate and the heifer was examined for accumulation of intrauterine fluid.

Approximately 24 h after presentation, the heifer developed a fever (40.1°C; normal range: 37.8°C to 39.2°C). Despite repeated transabdominal ultrasonic examinations, neither fetal movement nor a fetal heart beat was evident. Soon after, the heifer’s body temperature gradually returned to normal without medical intervention. Body weight, girth, and fetal viability continued to be assessed twice daily. Over the next 3 d, the heifer gained weight consistently and rapidly. Approximately 60 h after the heifer had become febrile, she began to produce milk and her teats and vulvar lips became edematous. A moderate amount of hemorrhagic to mucoid discharge was intermittently expelled from her vulvar lips. On vaginal examination, the birth canal appeared to be moderately relaxed. Because the heifer appeared to be aborting, the PRID device was removed. A serum sample taken at this time revealed a progesterone concentration of 3.3 ng/mL. That afternoon the heifer became recumbent, strained intermittently and released a large amount of fluid from her vulva. Due to the dam’s low value as a recipient and because of the apparent death of the fetus, the heifer was euthanized using an overdose of barbiturates.

Shortly before abortion, the total body weight of the dam had been 613 kg. At postmortem, her body weight was 547 kg. This indicated that a volume of approximately 66 L (66 kg) of fluid had been lost. Postmortem examination revealed that this was entirely allantoic fluid. There was marked subcutaneous edema of the ventral abdomen and mammary gland, composed of yellow-tinged gelatinous fluid.

The reproductive tract was ligated at the level of the cranial vagina for evaluation. The gravid uterus was markedly distended with fluid and was consistent with the size of a uterus at term. The fetal membranes showed thickening and edema. In some areas, the allantochorion was almost 1-cm thick and showed obvious pitting edema. Samples of allantoic fluid were collected and submitted for biochemical evaluation.

Placentomes were infrequent, with fewer numbers noted in the region of the internal cervical os. In total, 59 placentomes were counted (normal range: 75 to 100). The placentomes varied greatly in size, some were as small as 0.5 cm in diameter while others were as large as 12 cm in diameter. Infrequently, undersized cotyledons were present in the absence of corresponding caruncles. Severe edema of the amniotic membrane was present; ~20 L (~20 kg) of amniotic fluid was collected (normal range: 2 to 8 kg at term). A small quantity of amniotic fluid (~5 L) remained in the membrane after complete drainage had been attempted. Therefore, an additional 5 kg was added to the 20 kg figure, giving a total of 25 kg of amnionic fluid. Samples of amniotic fluid were collected and submitted for evaluation.

Since fluid was lost due to the ruptured allantois, the total volume of the allantoic fluid was determined as follows. We determined the sum of the following: the weight of the dam’s viscera (102.5 kg), the fetus (56 kg), the approximate weight of the amniotic fluid (25 kg), and the heifer’s carcass including her uterus and placenta (344.5 kg), giving a total weight of 528 kg. By subtracting this figure from the pre-abortion weight of the heifer (613 kg), the total volume of allantoic fluid was estimated to be 85 L (normal range: 4 to 15 L at term). A single, 56-kg, female fetus (normal weight at 7 months gestation: 8 to 18 kg), measuring 76 cm from crown to rump (length just outside normal range) was present in the uterus. The fetus was in posterior presentation with bilateral hip flexion, consistent with a breeched posture. The fetus was stained with meconium, presumably due to in utero respiratory distress. Marked subcutaneous edema was present in the fetus and there was bicavitary effusion. The fetal liver had a reticular pattern and it was enlarged, rounded, and diffusely pale. The fetal heart possessed a moderately enlarged left auricle relative to the size of the right auricle. On histopathology, prominent autolytic changes were evident within the chorioallantois and placentomes. There was abundant fibrovascular stroma within the villi of the caruncles. Many of the epithelial cells lining the thickened villi and corresponding cotyledonary crypts were hypotrophic and hypereosinophilic with pyknotic to karyorrhectic nuclei, consistent with necrosis. The fetal liver showed a moderate amount of tissue autolysis and marked expansion of the perisinusoidal space due to fluid accumulation.

Discussion

The gross appearance, histological findings, and results of diagnostic tests were consistent with hydrops allantois and amnion. Similar pathology has been described in other calves that were conceived by in-vitro fertilization (IVF) (1,2). Therefore, we concluded that our findings were likely related to the IVF process.

In-vitro fertilization and cloning constitute two of the most significant advancements in modern dairy and beef production systems. In-vitro produced (IVP) embryos can enhance crossbreeding schemes, genetic selection strategies, and become integrated into reproductive management programs (3). In this case, IVF was initiated after repeated attempts at embryo collection from the donor animal had been unsuccessful.

While IVP embryos may serve as an infinite economic resource, this technique is currently utilized by only a minute portion of the cattle industry worldwide. Serious complications are possible when either IVF or cloning procedures are used. Documented problems include prolonged gestation, suboptimal embryonic and fetal survival, abnormal placental development (placentomegaly, hydroallantois, and edematous placentomes in reduced numbers), and abnormal offspring development (increased fetal birth weights and the so-called large offspring syndrome) (3–6). Anomalies in gene expression, chromosome content, and an increased occurrence of apoptosis have also been recognized (3).

In one study, the percentage of calves with hydroallantois was 3.2% and 0.7% in IVF and artificially inseminated (AI) calves, respectively (5). In-vitro fertilized calves showed, on average, a 3-day prolonged gestation, a 10% increase in birth weight, more difficult calving, and a 2.4% greater incidence of perinatal mortality compared with AI calves (5). Also, cloned calves appear to have a higher incidence of neonatal mortality compared with normal calves or calves derived by IVF (6). An increased incidence of abnormal spinal cord development and asynchronous growth of fetal organs and limbs has also been documented (5,7).

In-vitro abnormalities may be due to dysfunctional oocytes, low oocyte recovery rates, poor oocyte quality, or may occur when certain culture systems are employed, such as co-culture with other cells or with the addition of biological additives to the culture media (4,8). Consequently, media alternatives are being investigated (8).

Large offspring syndrome (LOS) commonly occurs during late gestation, though the exact etiology remains uncertain (6). Interaction between in-vitro culture media and the early embryo, asynchronies between the uterus of the dam and the date of implantation, mitochondrial DNA interactions, and mutation during cell division are all potential causes (6).

Placental insufficiency characterized by a decreased number of placentomes and poor vascularization has been frequently documented during the first trimester in pregnancies involving IVF and cloned calves (1). Poor placentation has been associated with a compensated overgrowth of the remaining placentomes. Fetal abnormalities such as deficient angiogenesis, cardiomegaly, and ascites have also been associated with IVF (1,2,6).

In these cases, hydrops is the most obvious maternal abnormality, with fetal death occurring during the third trimester of pregnancy. Fluid accumulation is rapid, involving both the amnionic and allantoic cavities (1,2). Although the highest incidence of hydrops has been documented in cloned calves, it also appears to be higher in IVF pregnancies than in those that follow natural mating; 1.8 and 0.07%, respectively (2). However, these findings are not consistent in the literature (1). Although there has been much speculation as to electrolyte imbalances that mediate hydrops and the fetal abnormalities that precede these, they remain poorly understood (2).

Hyperactive fetal movement and hyperechoic allantoic and amniotic fluids are possible signs of fetal distress, but assessment of fetal movement is limited in cows due to abdominal depth and large fetal size which prevent complete visualization during ultrasonography (6).

There appears to be not only a growing interest in the use of in-vitro produced embryos but increased speculation regarding the efficacy, safety, and sustainability of this technique in the cattle industry. It has been suggested that limitations surrounding IVF can be overcome through further research with emphasis on the influence of the oocyte’s environment, the ovarian follicle, and culture conditions of embryos (3).