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The objective of this study was to determine effects of subcutaneous (s.c.) infusions of Ca at calving day on serum concentrations of Ca, P, Mg, β-hydroxybutyrate (BHB), glucose, body condition score (BCS), milk yield, and health in fresh cows receiving a dietary cation-anion difference diet during the dry period. Three hundred seventy-five fresh Holstein cows were blocked based on parity (143 cows in first parity, 108 cows in second parity, and 124 cows in third or greater parity) and randomly assigned to 1 of 4 experimental treatments immediately after calving. Treatments were control group without infusion (control; n=190); 1 s.c. infusion of 250mL of 40% Ca borogluconate (1SC; n=72) at calving; 1 s.c. infusion of 500mL of 40% Ca borogluconate (1SC n=63) at calving; 2 s.c. infusions of 250mL of 40% Ca borogluconate, one immediately after calving and the second 12 to 18h after first infusion (2SC n=50). Blood samples were collected immediately after parturition and at 1, 2, 4, and 7d in milk (DIM) for Ca, P, and Mg determination. Milk production, milk composition, and somatic cell count were recorded monthly up to 90 DIM. The evaluation of BCS was performed at calving and at 38 DIM. A subset of 9 cows per treatment group was randomly chosen to measure serum concentration of glucose and BHB at 2, 4, and 7 DIM. Total serum Ca in 1SC (8.95mg/dL), 1SC (9.27mg/dL), and 2SC (9.07mg/dL) was greater during the first week postpartum compared with control (8.45mg/dL). Serum concentrations of P, Mg, BHB, glucose, and milk yield were not affected by treatments. The dry matter intake during the first 24h after calving was higher for treatments 1SC (13.5kg), 1SC (15.0kg), and 2SC (15.6kg) relative to control (12.5kg). Milk somatic cell counts were lower for 1SC (90.5 cells/mL) and 2SC (82.2 cells/mL) than control (132.8 cells/mL). Risk ratio was >1 for development of metritis, and clinical and subclinical endometritis in control cows relative to 2SC cows, which indicates a higher risk of developing disease for control cows (2.03, 1.7, and 1.8 times, respectively). These results suggest that prophylactic s.c. infusions of Ca at calving can improve postpartum Ca status in Holstein cows and intake at 1 DIM. Given the reduction of metritis, clinical and subclinical endometritis, and subclinical hypocalcemia with treatment, the effect of s.c. Ca supplementation on immune status warrants further investigation.

STUDY QUESTION Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? SUMMARY ANSWER Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. WHAT IS KNOWN ALREADY MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. STUDY DESIGN, SIZE, DURATION This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. MAIN RESULTS AND THE ROLE OF CHANCE The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. LARGE SCALE DATA We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. LIMITATIONS, REASONS FOR CAUTION Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. WIDER IMPLICATIONS OF THE FINDINGS Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. STUDY FUNDING/COMPETING INTEREST(S) This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A