Twin management in the mare: A review.

Twin gestation in the mare is undesirable and can have disastrous consequences. As in many cases, the key to success in twin management lies in a thorough follow-up and accurate recording of clinical findings in the pre-breeding examination. A pregnancy diagnosis in the mobility phase is imperative for a good outcome in the event of twin reduction. If a twin gestation is not diagnosed during this early pregnancy stage, several other procedures exist for managing post-fixation twins (>16 days) with varying degrees of success. Most twin pregnancies are the result of multiple ovulations (dizygotic twins). However, monozygotic twins are also sporadically diagnosed, due to the increasing number of transferred in vitro produced equine embryos. In these cases, the most optimal treatment strategy still needs to be determined. This review provides an overview of the various twin reduction techniques described with the expected prognosis as well as of some less reported techniques with their results. In addition, physiological events and the reduction techniques are demonstrated to the user in virtual 3-dimensional illustrations.

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

BACKGROUND: Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. OBJECTIVES: To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. STUDY DESIGN: Retrospective case series. METHODS: Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. RESULTS: TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. MAIN LIMITATIONS: Missing retrospective data despite extensive follow-up. CONCLUSION: This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

Ultrasound-guided fetal thorax compression to reduce post-fixation twins in the mare.

BACKGROUND: Management of twin pregnancy after conceptus vesicle fixation in the horse is challenging because the reduction techniques described are either invasive, difficult to perform or associated with disappointing success rates. OBJECTIVES: To evaluate the success of transrectal ultrasound-guided fetal thorax compression for reducing post-fixation twin pregnancy in mares. STUDY DESIGN: Retrospective clinical study. METHODS: Sixteen mares were presented for twin reduction between 51 and 79 days of gestation. History obtained from the owner and/or referring veterinarian detailed information regarding the mare (age, breed), pregnancy (day of gestation, dizygotic versus monozygotic twins, unilateral versus bilateral fixation), treatment and outcome (one live fetus at discharge; live singleton at foaling) after twin reduction. Transrectal fetal thorax compression was performed under ultrasound guidance by two experienced operators. RESULTS: Overall 9 of 16 twin pregnancies were successfully reduced and the likelihood of success was significantly higher in dizygotic than monozygotic twins. The procedure was successful in 9 of 10 dizygotic twins but unsuccessful in all six cases of monozygotic twins. Among the dizygotic twins, two mares lost the pregnancy after discharge from the clinic, seven mares delivered a healthy foal of normal size. MAIN LIMITATIONS: Small case number. CONCLUSIONS: Transrectal ultrasound-guided fetal thorax compression is a minimally-invasive and successful technique for reducing dizygotic twin pregnancies at approximately 2 months of gestation, but does not lead to any live births in cases of monozygotic twins.

Twin reduction in the late embryonic period prevents the condition of freemartin in dairy cattle.

The condition of freemartinism occurs in the bovine heterosexual twin foetuses and refers to the resulting infertile female. Vascular anastomoses of the foetal membranes are the major reason of this anomaly. This study examines whether single born heifers following induced twin reduction at 28-34 days of pregnancy could develop normal reproductive functions. The study population derived from 367 lactating dairy cows carrying: unilateral twins (n = 178), bilateral twins (n = 174) or triplets (n = 15), in which manual embryo reduction was performed. The final study population was constituted of 95 single born twin females that reached 12 months of life and entered into the AI period. Of these heifers, 40 have had one unilateral co-twin, 49 one bilateral co-twin, and 6 two contralateral co-twins. A total of 1688 heifers inseminated during the same period were used as controls to compare the rates of pregnancy at 15 months of age, culling due to infertility and birth before the age of 24 months. No differences were detected between groups. With an accepted incidence rate of 50% heterosexuality for all twin sets, we should assume that half of our study population had a male co-twin. Our results indicate lack of effect of the male co-twin on normal development of the gonads and genital tract of his female partner during the late embryonic period.

The effect of embryo reduction and transfer on luteostasis in the mare.

This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulation resulted in luteostasis in 78%, 87.5%, 78% and 37.5% of mares. Only 22%, 37%, 0% and 12%, respectively, of these mares remained pregnant. In the Day 10, 11 and 12 recipients luteostasis occurred on at least one occasion when an embryo was detected at 24 h but not at 48 h post transfer. In the Day 12 recipients luteostasis occurred on three occasions (3/9;33%) when the transferred embryo was not detected at 24 h. In Experiment 3 reduction of a Day 11 embryo 24 h after transfer to a Day 10 (n = 4), 11 (n = 6), 12 (n = 6) or 13 (n = 6) recipient resulted in luteostasis in 100%, 83%, 100%, and 83% of mares. All five Day 11 recipients that had an embryo reduced 12 h post transfer became luteostatic. These results suggest there is plasticity overall, but individual rigidity, in the timing of maternal recognition of pregnancy. Furthermore, an intact embryo need only be present in the uterus for 12 h to cause luteostasis.

A retrospective study of twin pregnancy management in mares.

The aim of the study was to compare three methods of reducing twin pregnancy in mares to maintain a single pregnancy. As multiple pregnancies in mare are always undesirable, early ultrasound diagnosis makes possible management of twin pregnancies and extra embryo removal. In years 2010-2018, 16494 mares were sonographically tested for early pregnancy, finding 868 cases of twins (471 bilateral and 397 unilateral). 260 mares with a confirmed bilateral tween pregnancy were subjected to manual crushing of one embryo and administration of flunixin at a dose of 1.1 mg/kg BW. 186 mares were subjected only to the embryo crushing procedure. 25 mares from this group were on a restrictive diet. In the unilateral twin pregnancy mare group, 62 were subjected to manual embryo reduction with simultaneous treatment with flunixin, 60 had only manual embryonic vesicle crush and 210 had a restrictive diet. Determination of success, measured as the development of a single pregnancy, were monitored 2 weeks after the procedure, between the 50th and 60th day of pregnancy and after the 90th day of pregnancy. In general, warm-blooded mares were more prone to a twin pregnancy, and at the same time, all the procedures used to reduce it to a single pregnancy caused a greater risk of losing both embryos than in the case of cold-blooded mares. The beneficial effect of administering flunixin after manual removal of one embryo on the maintenance of the other has been experimentally proven in both unilateral and bilateral twin pregnancy.

Transfer of a single embryo versus drainage of subordinate follicles to prevent twin pregnancies in dairy cows. Why not both?

In this study, we present two proposed approaches to prevent twin pregnancies in dairy cattle: 1) single, in vitro-produced embryonic transfer into a recipient cow or 2) subordinate follicle drainage at the time of insemination. Both procedures lead to improved embryonic survival. As the use of sexed semen generates herd replacements and additional heifers, we propose the transfer of a single female cattle embryo into cows that are not suitable for producing replacements, and follicular drainage in lactating cows with genetic merit. This should eliminate economic losses associated with twin pregnancies and increase cattle output of the herd.

Abortion in dairy cattle with advanced twin pregnancies: Incidence and timing.

This study examines incidences and timing of abortion in Holstein Friesian dairy cows with advanced twin pregnancies in north-eastern Spain. The study population consisted of 1,194 twin pregnancies in two herds recorded from 2010 to 2015: 522 bilateral and 672 unilateral. The presence of live twins was determined by transrectal ultrasonography between 55 and 61 days post-AI and pregnancy confirmed 60 days later. Abortion was recorded in 278 (23.3%) cows before Day 260 of pregnancy: 7 (1.3%) in bilateral and 271 (40.3%) in unilateral twin pregnancies. Using binary logistic regression procedures the following were excluded as risk factors for abortion: herd, year, heat stress (temperature-humidity index values >72), lactation number and right versus left side for unilateral twins. Based on odds ratios, cows carrying unilateral twins were 49.9 (right unilateral twins) and 49.3 (left unilateral twins) times more likely to suffer abortion than cows with bilateral twins. The average time of abortion for unilateral twins was 173 ± 32 days and ranged from 135 to 249 days, 43% of abortions occurring between 135 and 154 days of gestation. The authors suggest prevention or reduction of unilateral twin pregnancies by follicular puncture and drainage of subordinate follicles at AI or manual rupture of the amniotic vesicle of one of the two twins at the time of pregnancy diagnosis.

Effects of mares' age and day of gestation on efficacy of transvaginal ultrasound-guided twin reduction.

OBJECTIVE: Transvaginal ultrasound-guided aspiration (TUA) is a procedure which can be used for the reduction of twins post-fixation in the mare. The aim of this study was to evaluate the effect of the age of mares and the day of gestation on the outcome of TUA treatment. MATERIAL AND METHODS: In 88 mares, diagnosed pregnant with twins, TUA of the yolk sac or allantoic fluid was performed between day 30 and 62 of gestation. Mares were aged 3-22 years. Ultrasonographic examination for a viable singleton pregnancy was performed by referring veterinarians 5-7 days and 4 weeks after TUA. Based on reported findings, effects of age and day of gestation on pregnancy rates were evaluated. RESULTS: Four weeks after TUA, 67% of the cases resulted in a viable singleton pregnancy. Five to 7 days after TUA treatment, the success rate was 74%. The gestational period did not affect the outcome, irrespective of the age of the mare. In contrast, success rates decreased with increasing age of the mares (84% ≤ 7 years vs. 67% 8-14 years vs. 57% ≥ 15 years). In mares aged 8-14 years, a decrease in singleton pregnancies was observed, if TUA was performed after day 35 of gestation. Success rates were slightly higher, if twin vesicles were localized within separate uterine horns (73%) as compared to the same horn (66%). Differences in singleton pregnancy rates were not statistically significant (p > 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: TUA was found to be an effective procedure for reduction of twin pregnancies performed at days 30-62 of gestation. Success rates for singleton pregnancies were high for young mares ≤ 7 years old (84%) and middle aged mares treated before day 36 of pregnancy (74%). Duration of pregnancy at the time of TUA did not have a major impact on the outcome. Nevertheless, the procedure should optimally be performed around days 32-35 of pregnancy to allow for the possibility of natural reduction before treatment and rebreeding in case of a total pregnancy loss after TUA.

Transabdominal ultrasound-guided multifetal pregnancy reduction in 10 cases of monkeys.

Intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) in nonhuman primates, e.g. rhesus and cynomolgus monkeys, has been widely used in researches of reproductive and developmental biology, and the success rate has been improved significantly. However, unwanted multiple pregnancy occurs frequently during the ICSI-ET in monkeys, most of which leads to miscarriages. To improve the birth rate of pregnancies and to safeguard health of host and baby monkeys, multifetal pregnancy reduction (MPR) is necessary. In this study, a total of 10 monkeys with multiple pregnancies received MPR through transabdominal ultrasound-guided potassium chloride injection into beating hearts of selective fetuses. To assess MPR efficiency, 31 monkeys with normal singleton pregnancies and 25 monkeys with twin pregnancies without MPR were used as controls. The aim of the reduction is to keep only one fetus, no matter twin or triplet pregnancy originally. Our results show that six cases of MPR were successful and all of them retained single fetus. Moreover, about 1 month (30.2 ± 1.2 days) of gestation is a better timing for MPR than later stage (50.7 ± 1.9 days). We also found that the remaining fetuses developed normally with full-term gestation and normal birth weight. In conclusion, transabdominal ultrasound-guided potassium chloride injection is a safe and effective MPR method for monkeys with multiple pregnancies.

Efficacy of transvaginal ultrasound-guided twin reduction in the mare by embryonic or fetal stabbing compared with yolk sac or allantoic fluid aspiration.

Transvaginal ultrasound-guided pregnancy reduction (TUGR) is a procedure described for the management of twins post-fixation in the horse. Success rates are often disappointing but are reported to be more favorable for bilaterally situated twins and when intervention takes place before day 35 of gestation. This study aimed to determine whether stabbing the embryo/fetus rather than aspirating conceptus fluids improved the likelihood of success, measured as the birth of a normal live singleton foal. Data from 103 TUGR interventions were analyzed by logistic regression analysis; method of treatment, relative conceptus location (i.e., uni- vs. bilateral), and stage of gestation were included as interdependent factors that potentially influence the outcome. Overall, 34/103 (33%) TUGR interventions resulted in a single live foal. There was no significant difference (P = 0.14) in the outcome between TUGR based on fetal stabbing (12/28: 42.9%) versus fluid aspiration (22/75: 29.3%). There was also no significant influence (P = 0.11) of the conceptuses being located unilaterally (19/65: 29.2%) versus bilaterally (15/38: 39.5%). However, TUGR was numerically more successful (P = 0.05) when performed ≤ Day 35 of gestation (21/53: 39.6%), as opposed to > Day 35 (13/50: 26%). Day 45 may represent an even more critical time point because only 2 out of 15 TUGRs (13.3%) performed beyond this day resulted in the birth of a live foal, compared with 11/35 (31.4%) performed between Days 36 and 45. Although the numbers are low, this suggests that TUGR is not the method of choice for reducing > Day 45 twins. Four pregnancy losses were recorded 1 to 7 months post-TUGR (4/38: 10.5%), and although it is tempting to attribute the losses to TUGR, this rate of late gestation pregnancy loss is normal. We conclude that TUGR by fetal stabbing does not offer significant advantages over fluid aspiration. However, TUGR should be performed before Day 35 of gestation and is considered primarily a salvage procedure to be used when re-breeding is not a viable alternative.

Evaluation of pregnancy and foaling rates after reduction of twin pregnancy via transvaginal ultrasound-guided aspiration in mares.

OBJECTIVE: To assess pregnancy and live foaling rates after reduction of twin pregnancy via transvaginal ultrasound-guided aspiration (TUA) in mares and evaluate effects of gestational period, localization of conceptuses, fluid aspiration volume, and combination of TUA with embryonic or fetal puncture on these outcomes. DESIGN: Clinical trial. ANIMALS: 44 mares pregnant with twins (25 to 62 days of gestation). PROCEDURES: TUA was performed in all mares and combined with embryonic or fetal puncture in 13. Follow-up ultrasonographic examinations were performed by referring veterinarians. Effects of gestational period and TUA-related variables on pregnancy and foaling rates were assessed. RESULTS: Singleton pregnancy (32/44 [73%]), persistent twin pregnancy (3/44 [7%]), or loss of both conceptuses (9/44 [20%]) was confirmed 5 to 7 days after TUA. Two mares with persistent twin pregnancy underwent another TUA, and 1 underwent prostaglandin F(2α)-induced abortion; these were excluded from subsequent analyses. Eighteen of 24 mares reevaluated 3 to 4 weeks after TUA were pregnant with 1 conceptus. Twenty of 41 (49%) mares delivered live singleton foals. Gestational period of TUA did not affect early pregnancy rates. Foaling rate for mares that underwent TUA after day 42 (0/5) was lowest of all groups and was significantly lower than that of mares that underwent TUA between days 31 and 35 (9/14). Three of 13 mares that underwent TUA with embryonic or fetal puncture delivered live foals, compared with 17 of 28 that underwent TUA alone. Effects of other variables were nonsignificant. CONCLUSIONS AND CLINICAL RELEVANCE: TUA was effective for reduction of twin pregnancy in mares, with best results achieved before gestational day 43.

Manual rupture versus transvaginal ultrasound-guided aspiration of allanto-amniotic fluid in multiple pregnancies: a clinical approach to embryo reduction in dairy cattle.

To avoid the problems associated with twinning in dairy cattle, one of the embryos may be eliminated. This study compares the effect on pregnancy maintenance of two embryo reduction techniques, manual rupture (MR) and transvaginal ultrasound-guided aspiration (TUGA) of allanto-amniotic fluid, in Holstein-Friesian cows with multiple pregnancies. In the first experiment, 61 lactating cows bearing unilateral twins (n=27), bilateral twins (n=30) or triplets/quadruplets (n=4) were subjected to MR (n=45) or TUGA using a 17-G neddle (n=16) on day 28-34 of gestation. In 21 and 10 cows undergoing MR and TUGA embryo reduction, respectively, pregnancy loss occurred before day 90 (46.7 vs. 62.5%, P= 0.28). Through binary logistic regression, the type of pregnancy was identified as the only variable significantly affecting pregnancy maintenance (P=0.03). Based on the odds ratio, the risk of pregnancy loss was 4.1 times higher for unilateral twins than for bilateral twins (70.4 vs. 36.7%, respectively, P=0.01). No effect was detected on pregnancy maintenance of the technique used (P=0.17) or of the interaction technique by type of pregnancy (P=0.22). In the second experiment, a 22-G needle was used to perform TUGA on 22 lactating cows. The pregnancy loss rates were 44.4% (4/9), 18.2% (2/11) and 50% (1/2) for cows bearing unilateral twins, bilateral twins and triplets, respectively. The total pregnancy loss rate following TUGA using the 22-G needle tended to be lower than that using the 17-G needle (31.8 vs. 62.5%; P=0.06). Our results suggest that TUGA using a 22-G needle could be the method of choice to perform embryo reduction in cows carrying multiple pregnancies.

Manual reduction of multiple embryos in the mare: the effect on subsequent pregnancy outcome.

To determine whether manually reduced multiple pregnancies (MPs) are at a greater risk of pregnancy loss than single pregnancy (SP) in mares, and to examine if a difference exists in the timing of pregnancy loss between manually reduced MPs and SPs, 1916 Thoroughbred mares were ultrasonically monitored every 2 days during oestrus to confirm ovulation, and up to Day 40 post-ovulation to confirm pregnancy. Ultimate pregnancy outcome was ascertained from the General Thoroughbred Stud book and classified as live foal (LF), early abortion (EA; Days 40-150), slipped foal (SF; Days 150-term), and barren (B; Time of pregnancy loss unknown). Significantly (P<0.05) more SPs failed (17.23%; 226/1312) than manually reduced MPs (13.41%; 81/604). Both SPs and MPs were at greatest risk of being lost as EA (72.16%; 127/176 and 61.67%; 37/60, respectively) compared to SF (27.84%; 49/176 and 38.33%; 23/60). There was no significant difference in the time of greatest risk of pregnancy loss between manually reduced MPs and SPs. It was concluded that owners and veterinarians can be assured that manual reduction of MPs does not increase the risk of pregnancy loss above those associated with SPs.

Clinical implications of induced twin reduction in dairy cattle.

Embryo reduction may prevent the negative effects of twinning in dairy cattle; however, the technique may carry an additional risk of pregnancy loss. The aim of this study was to evaluate the effect on pregnancy maintenance of embryo reduction by manual amnion rupture in unilateral and bilateral twin pregnant cows. A secondary objective was to examine the dynamics of endocrine factors following the treatment. On Day 35-41 of gestation 55 cows bearing two live twin embryos (28 bilateral, 27 unilateral) were randomly assigned to a twin reduction group (n = 27; cows fitted with a progesterone releasing intra-vaginal device for 21 days after manual amnion rupture) or control group (n = 28; untreated cows). Pregnancy loss before Day 90 was recorded in nine control and eleven twin reduction cows (32.1% vs 40.7%, respectively, p = 0.508). Logistic regression models indicated that laterality was the only variable significantly affecting pregnancy loss. The pregnancy loss risk was 8.7 times higher for unilateral than for bilateral twin pregnancies (59.3% vs 14.3%, respectively, P < 0.001) yet was similar in the unilateral control and unilateral twin reduction cows (62.3% vs 53.8%, respectively, P = 0.581). In contrast, four of 14 cows with bilateral twin pregnancies undergoing twin reduction lost their pregnancies while no losses were recorded in control cows with bilateral pregnancies (P = 0.049). A rise in plasma progesterone concentration was detected on the day following treatment in the twin reduction group and concentrations remained high within the first week of treatment. Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations fell between Day 35-41 and Day 42-48, regardless of treatment. Our findings indicate that embryo reduction by manual amnion rupture did not carry an additional risk of pregnancy loss for unilateral twin pregnancies, whereas it increased the risk of pregnancy failure in bilateral twin pregnancies. However, benefits of preventing cows from delivering twins might also be considered when assessing the success of embryo reduction in bilateral twin pregnancies.

Transvaginal ultrasound-guided aspiration of unilateral twin gestation in the mare.

Embryo reduction of unilateral twin vesicles is normally successful before fixation of the vesicles. After fixation, however, it becomes a challenge, and the later in the gestation period that the reduction is performed, the greater the challenge. One therapy to reduce a twin into a singleton pregnancy is the transvaginal ultrasound-guided aspiration (TUGA) of one of the vesicles. The results of 35 unilateral twin reductions by TUGA are discussed in this paper and possible negative influences of age, parity and days pregnant are analysed statistically.

Twin pregnancy experimental model for transvaginal ultrasound-guided twin reduction in mares.

Multiple pregnancies are still an important cause of noninfectious abortion, stillbirth, neonatal mortality, and significant delays in reproductive performance in mares. Despite new management techniques, reduction in multiple pregnancies is an ongoing preoccupation and challenge for the equine veterinarian. The aim of the present study was to establish a twin pregnancy experimental model in the mare to study the effectiveness of a transvaginal ultrasound-guided embryonic vesicle injection. Mares in heat were inseminated and then received an embryo at day 7 of the estrous cycle. At days 14 and 30, 53.5% (n = 23) and 23% (n = 10) of the mares, respectively, were carrying twins. Twin pregnancies were reduced at day 30 by transvaginal ultrasound-guided puncture of the embryonic vesicle (control, n = 5) or by transvaginal ultrasound-guided injection (TVUEVI) of 25 mg of amikacin into the embryonic vesicle (n = 5). The TVUEVI treatment had a 40% success rate and no significant variations in progesterone and prostaglandin metabolite were observed. Even though the technique does not seem very effective, the experimental model could be useful for clinical research in embryo reduction and early embryonic loss.

Management of postfixation twins in mares.

Methods to manage twins after fixation include natural reduction,dietary reduction, transvaginal ultrasound-guided aspiration,surgical removal, craniocervical dislocation, and transabdominal ultrasound-guided injection. Of these, results have been inconsistent with regard to producing a single healthy foal, except for craniocervical dislocation. This new technique enables the twin to be reduced before complete placenta formation has occurred, allowing the remaining fetus to use the entire endometrial surface and grow to its full potential.