Early embryonic mortality in bovine with emphasis on heat stress and its amelioration

Introduction

The dairy sector of India represents the single largest agricultural commodity in the country, comprising 193.46 million cattle and contributing nearly 5.5% to the national GDP. India is the world's largest milk producer, with a recorded output of 230.6 million tonnes during 2022-23. Sustaining this scale of output has relied on intensive genetic selection for high milk yield, and this selection pressure has progressively been accompanied by a decline in reproductive efficiency (Bello et al., 2012) [4] . In lactating cows, the heightened metabolic demand further downregulates expression of the DKK1 gene a regulator of neural development, glucose homeostasis, and embryonic competence (Denicol et al., 2014) [15] . These reproductive failures manifest most visibly as embryonic mortality, a major source of economic loss to livestock farmers. In cattle, the fertilization rate is approximately 90%, whereas the average calving rate is only about 55%, yielding an overall embryonic/fetal mortality of nearly 35%, with 70-80% of these losses occurring between Days 8 and 16 post-insemination (Diskin et al., 2006) [17] . Early embryonic loss is consequently more pronounced in high-yielding dairy cattle and remains a primary constraint to milk-production efficiency. Although late embryonic mortality accounts for less than 10% of pregnancy loss, heat stress alone may contribute an additional 15-20% absolute increase in embryonic mortality, making HS a critical determinant of fertility in tropical and subtropical environments.

Early embryonic development and pregnancy recognition

In cattle, Day 0 corresponds to estrus, with ovulation occurring approximately 18 h later.

Ovulation induces luteinization of granulosa and theca cells to form the corpus luteum, the primary source of progesterone (P4), an essential hormone for early embryo survival. P4 synthesis draws on cholesterol from three sources: de novo synthesis, hydrolysis of cholesterol esters, and uptake of circulating lipoproteins. Cholesterol is transported intracellularly by sterol carrier protein-2, and its transfer to the inner mitochondrial membrane the rate-limiting step in steroidogenesis is mediated by steroidogenic acute regulatory protein. Cholesterol is then converted to pregnenolone by CYP11A1 and subsequently to P4 by 3β-hydroxysteroid dehydrogenase (3β-HSD) (Seedorf et al., 2000) [59] . Around Days 16-17 of pregnancy, maternal recognition of pregnancy is established through IFN-τ secreted by the elongating conceptus. IFN-τ suppresses endometrial estrogen receptor expression, preventing oxytocin receptor upregulation and prostaglandin F₂α (PGF₂α) release, thereby protecting the CL from luteolysis and maintaining P4 secretion. IFN-τ also induces interferon-stimulated genes and downregulates endothelin-1, transforming growth factor-β1, serpin-1, and thrombospondins 1 and 2, collectively supporting luteal maintenance and pregnancy establishment (Bott et al., 2010) [7] .

Causes of early embryonic mortality 3.1 Genetic and Chromosomal Factors

Genetic abnormalities account for approximately 10% of embryonic losses during the first two weeks of gestation and may contribute up to 20% of total embryonic and fetal mortality (Vanroose et al., 2000; Diskin & Morris, 2008; Figure 1) [16,68] . Deficiency of uridine monophosphate synthase results in embryonic death between Days 40 and 50 of gestation (Robinson et al., 1984), and gross chromosomal abnormalities account for about 5% of embryonic mortality (Peters, 1996) [48] . Robertsonian translocation (1/29), which fuses chromosomes 1 and 29 to yield a karyotype of 59 chromosomes (Gustavsson, 1979) [19] , is associated with embryonic death during the first or second week of gestation. Dysregulated expression of oxidative stress-related genes upregulation of phospholipase A2 and glutathione peroxidase along with downregulation of annexin A1 and lactoferrin disrupts oxidative balance and meiotic competence, predisposing embryos to early loss (Puglisi et al., 2013) [51] .

Endocrine factors

Endocrine dysfunction is a leading cause of EEM. Inadequate preovulatory follicular development produces luteal insufficiency via delayed CL development, premature luteolysis, or functional luteal defects, leading to inadequate P4 secretion required for pregnancy maintenance. Suboptimal P4 during the first week after ovulation impairs uterine receptivity by altering MUC1 expression and compromises implantation. Conversely, suprabasal P4 (>1 ng/ml) before ovulation suppresses LH pulsatility, delays the LH surge and ovulation, and promotes follicular overmaturation and embryo-uterine asynchrony, producing aged gametes, repeat breeding, and EEM (Kimura et al., 1987) [31] . High-yielding dairy cows also exhibit reduced peripheral P4 because of increased hepatic metabolism, predisposing them to embryonic loss. Luteal dysfunction contributes to conception failure in approximately 67% of buffaloes and 65% of crossbred cattle (Jisna, 2024; Nivetha, 2024) [28,45] . Cows with estrous P4 concentrations >1 ng/ml show a lower ovulation rate than those with <0.5 ng/ml (Burns et al., 2008) [8] , while prolonged CL lifespan has been associated with EEM, abortion, and fetal mummification (Unuane et al., 2011) [67] .

Nutritional factors

Nutritional imbalance contributes to EEM through endocrine dysfunction, oxidative stress, and impaired metabolic homeostasis. Affected cows exhibit reduced P4 and estradiol-17β concentrations together with altered protein, urea, creatinine, cholesterol, and liver-enzyme profiles and elevated endogenous intoxication indices, reflecting hepatic and renal dysfunction. Embryonic loss is further associated with deficiencies of zinc, copper, manganese, selenium, magnesium, iodine, and vitamins E and C, and with reduced glutathione peroxidase and catalase activity each undermining antioxidant defence, promoting lipid peroxidation, and amplifying oxidative stress (Nezhdanov et al., 2017) [42] .

Infectious factors

Infectious agents contribute substantially to EEM; bacterial infections (14.49%) are more prevalent than viral (10.57%) and fungal (5.31%) infections in abortus and stillbirth submissions. Among viral pathogens, bovine herpesvirus-1 and bovine viral diarrhea virus are most frequently reported, while common bacterial pathogens include Trueperella pyogenes, Bacillus spp., Listeria spp., Escherichia coli, and Leptospira interrogans (Givens & Marley, 2008; Kumar et al., 2011; Gates et al., 2013) [21,33] .

immunological factors

Successful embryo survival requires maternal immune tolerance toward the semi-allogeneic conceptus. During early pregnancy, a shift from Th1-to Th2-dominant immunity, together with endometrial and placental expression of insulinlike growth factor-1, IGF-2, and IGF-binding proteins, facilitates nutrient partitioning and conceptus development (Robinson et al., 2000; Sferruzzi-Perri et al., 2007) [56,60] . IFN-τ secreted by the conceptus suppresses lymphocyte proliferation, protecting the embryo from maternal immune rejection (Skopets et al., 1992) [61] . Elevated IGFBP-1 and IGFBP-4 concentrations, together with reduced IL-6, IL-13, and IFN-τ levels, have been associated with early embryonic and fetal death (Sobhy et al., 2021) [62] . [21,33] 4. Predisposing factors for eem Species. EEM is generally higher in buffaloes than in cattle because of greater susceptibility to heat stress and associated endocrine disturbances (Kumbhar et al., 2021) [35] .

Schematic Overview of Pathogens Affecting Reproduction

Breed. High-producing dairy breeds, particularly Holstein cows, exhibit a higher incidence of EEM than dual-purpose breeds such as Simmental and Brown Swiss. Crossbred cattle likewise show a higher prevalence of repeat breeding (17.57%) than indigenous breeds (8.64%) (Verma et al., 2018) [69] .

Milk yield. High milk production increases metabolic demand, predisposing dairy cows and buffaloes to negative energy balance and a greater risk of early embryonic loss (Kaneda et al., 2024) [29] .

Parity. Embryonic mortality generally increases with advancing parity. Swedish Red and Swedish Holstein cows in their third or later parity exhibit greater fetal loss (>61 days after AI) than younger cows (Nyman et al., 2018) [46] . However, primiparous cows may also experience higher EEM because of reproductive immaturity and incomplete metabolic adaptation.

Age. Maximum conception rates are achieved in heifers bred at 15-16 months of age, whereas first breeding after 26 months reduces pregnancy per insemination by approximately 13% (Martin et al., 2015) [38] . Higher EEM has also been reported in cows aged 2.0-2.4 years, while cows aged 3.5-4 years exhibit the lowest losses (Puklová et al., 2014).

Body Condition Score and energy balance. Cows with suboptimal BCS (<2.5 or 2.75-3.5) experience greater embryonic loss than those with optimal BCS (>3.75) (Senosy et al., 2012) [65] . During early lactation, peak milk yield precedes peak feed intake by several weeks, producing NEB, mobilization of body reserves, and approximately a one-unit loss in BCS each compounding the risk of EEM (Parmar et al., 2016) [47] .

Early detection of embryonic mortality 5.1 Preimplantation factor

• Secreted by trophoblast cells and placental tissue (Barnea, 2007; Stamatkin et al., 2011) [3,64] . • Peptide sequence: MVRIKPGSANKPSDD.

• Detection method: PIF ELISA.

• Enhances endometrial receptivity, promotes trophoblast invasion, and modulates immunity. • Detectable in serum at 91.3% by Day 10 and ~100% by Day 20 of pregnancy.

Progesterone (P4) assay in blood

• Low or declining P4 between Days 10 and 20 postinseminations is associated with higher embryonic loss in buffalo and cattle (Campanile et al., 2005) [50] . • On Day 21, P4 <2 ng/ml indicates non-pregnancy and implantation failure (Roa et al., 1998) [54] . • Cutoff serum P4 concentrations of >0.95 ng/mL on Day 0, <1.86 ng/mL on Day 5, and <1.98 ng/mL on Day 18 of the estrous cycle predict luteal phase deficiency in buffaloes owing to suprabasal, delayed rise, and early fall of P4, respectively. • Milk P4 >5 ng/mL on Days 21-24 is consistent with pregnancy (Humblot,2001) [25] .

Interferon-stimulated genes

• Detected in blood on Days 18-20.

• Expression correlates with MX2 and ISG15 mRNA levels in peripheral blood leucocytes. • Particularly accurate for nulliparous cows.

IFN-τ in cervical fluid

• Detected by ELISA with a limit of detection of ~54 pg/mL.

• Optimal detection window: Days 16-19 after AI.

• Detection of IFN-τ on Day 17 followed by ultrasonographic pregnancy loss on Day 32 identifies loss attributable to EEM. • Sensitivity ~80%; specificity ~75% (Bishop et al., 2025) [6] . [71] ; Zoli et al., 1992b [72] ; Sasser & Ruder, 1987 [73] Pregnancy [13,20] . The resulting increase in maternal body temperature disrupts ovarian function, uterine receptivity, fertilization, and embryonic development, making HS a major cause of EEM (Berman, 1985; De Rensis & Scaramuzzi, 2003) [5,12] .

Comparative Profile of Pregnancy-Associated Biomarkers

Neuroendocrine effects of HS

The physiological response to HS is mediated principally through activation of the HPA axis and the sympatheticadrenal-medullary axis. HS stimulates hypothalamic release of corticotropin-releasing hormone, leading to secretion of adrenocorticotropic hormone from the anterior pituitary and consequent cortisol release from the adrenal cortex. Although cortisol supports metabolic adaptation by promoting gluconeogenesis and energy mobilization (Ayoob et al., 2018) [2] , sustained elevation suppresses reproductive function by inhibiting KNDy neurons and consequently reducing GnRH, LH, and FSH secretion. This endocrine disruption impairs follicular development, steroidogenesis, ovulation, oocyte competence, and fertilization, collectively increasing EEM (Khan et al., 2020) [30] . HS also reduces inhibin secretion and alters follicular dynamics, further compromising fertility. Elevated cortisol concentrations in plasma, milk, and faecal 11,17-dioxoandrostane have been proposed as reliable biomarkers of HS in cattle (Möstl et al., 2002) [41] . [66] . Disruption of NF-κB signalling further impairs cell survival and embryonic development under thermal stress.

Immunological effects of hs

Lymphocytes: HS and cortisol suppress T-cell activation by inhibiting NF-κB and MAPK signalling, reducing lymphocyte proliferation and adaptive immune function. Although the magnitude of lymphocyte changes varies among breeds, thermotolerant indigenous cattle generally exhibit less immune suppression than high-producing exotic breeds (Elvinger et al., 1991) [19] .

Transcriptomic response to HS in cattle embryos

HS induces profound transcriptomic and epigenetic alterations in bovine embryos, initially as adaptive responses but ultimately as stress persists impairing embryonic development. Thermal stress activates chromatin remodelling through histone acetylation and demethylation, leading to activation of stress-responsive transcription factors and increased expression of heat-shock protein genes, particularly HSP70. During early embryogenesis, development depends on maternally derived mRNA and protein until embryonic genome activation at the 8-16-cell stage. This transition involves extensive DNA methylation reprogramming mediated by DNA methyltransferases, rendering embryos highly susceptible to heat-induced epigenetic disruption (Jiang et al., 2014; Dobbs et al., 2013) [18,27] . Consequently, HS alters gene expression, compromises embryo quality, and reduces developmental competence (de Barros & Paula-Lopes, 2018) [14] . Heat stress enhances acetylation of the HSP70 promoter, increasing HSP70 expression, whereas embryos derived from heat-stressed oocytes accumulate the repressive histone mark H3K9me3 and heterochromatin protein-1, producing chromatin condensation and apoptosis (Kisliouk et al., 2017; Camargo et al., 2019b) [9,10,32] . Although HSP70 protects embryos by preventing protein misfolding and inhibiting apoptosis, prolonged or severe HS overwhelms these protective mechanisms (Mosser et al., 2000). Other thermotolerance-associated genes, including HSPA14 and other HSP family members, exhibit stage-dependent responses to heat exposure (Sakatani et al., 2012; Hassan et al., 2019) [23,58] . Heat stress also disrupts expression of genes essential for embryonic development, including DNMT1, CDH1, and Cx43, as well as stress-responsive genes (HSPB11, HSPBP1, AKR7A2, GSTA4) and components of the ubiquitinproteasome pathway, reflecting enhanced protein-quality control under thermal stress (Sakatani et al., 2013) [57] . Reduced expression of developmental genes such as AQP3 and ATP1A1 further impairs blastocyst expansion and developmental competence, contributing to increased apoptosis and EEM (Camargo et al., 2019a) [9,10] . Overall, HS triggers cytoprotective transcriptomic responses, yet persistent thermal stress disrupts epigenetic programming and developmental gene expression, ultimately compromising embryo viability and pregnancy establishment.

Ameliorative strategies for heat stress-associated EEM 7.1 Managemental strategies

• HS should first be minimized through environmental management provision of shade, improved ventilation, evaporative cooling systems (fans, sprinklers, misters), and continuous access to clean drinking water. • RTLS for monitoring movement and activity.

• GPS for tracking grazing animals.

• VF for controlled grazing and movement management.

• RR as an early indicator of thermal stress.

• CBT for assessment of internal thermal load.

• ST measured by Infrared Thermography.

• TT, VT, RT as body temperature indicators.

• IoT integration of sensors with automated cooling systems for real-time HS management.

Hormonal therapy Gonadotropin-releasing hormon

• 250 µg GnRH at the time of AI increased pregnancy rate by 22.5% in repeat-breeder cattle (Morgan and lean, 1993) [40] . • 200 µg GnRH on Day 20 post-AI reduced embryonic mortality in buffaloes (Abulaiti et al., 2023) [1] .

• GnRH enhances luteal function and increases P4 secretion, improving embryo survival.

Human chorionic gonadotropin (HCG)

• hCG on Day 6 post-AI increased pregnancy rate to 67.5% (Nishigai et al., 2002) [43] . • 1000 IU hCG post-AI induces luteinization of the dominant follicle and accessory CL formation in cattle (Cooper et al., 1991) [11] .

Progesterone (P4): P4 supplementation during early pregnancy supports embryo development, although timing is critical. PRID insertion on Day 5 post-AI reduced pregnancy rate and increased EEM because endogenous CL output declined after device removal (Villarroel et al., 2004) [70] . [39,44,63] . • Unlike interferon-α, recombinant IFN-τ does not induce hyperthermia (Plante et al., 1991) [49] .

Kisspeptin

Cyclooxygenase Inhibitors

• Meloxicam (0.5 mg/kg IM) on Days 13-15 post-AI reduced prostaglandin synthesis and increased conception rate by approximately 20% (Rajkumar et al., 2010) [53] .

Immunomodulators

• Escherichia coli LPS (100 µg, intrauterine) stimulates the uterine immune response (Dash et al., 2016). • Carvacrol (0.1%) + Lugol's iodine (0.1%), 50 mL intrauterine, improves uterine health (Lehimcioglu et al., 2019) [37] . • Neem-oil extract (30-40 mL, intrauterine) yielded a 71.4% conception rate (Kumar et al., 2013) [34] .

Treatment Strategies for Infectious Causes of EEM

• Intrauterine antibiotic therapy: oxytetracycline; levofloxacin + ornidazole/metronidazole suspension. • Hormonal therapy: cloprostenol (500 µg IM); dinoprost (25 mg IM). • Combined hormonal + antibiotic therapy: oxytetracycline (1500 mg intrauterine) + cloprostenol (500 µg IM).

• Pre-and post-AI preventive therapy: penicillin G (10 lakh IU in 20-30 mL distilled water), administered 5-6 h before and 3-6 h after AI; streptomycin-penicillin (1 g in 20-30 mL distilled water), on the same schedule. • Systemic antibiotic therapy on Day 4 post-AI (embryo entry into uterus) and Days 9-10 post-AI (zona-pellucida hatching stage) reduces uterine infection and improves embryo survival in high-risk animals.

Conclusion

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