Metabolic signature of cervical mucus in ewe breeds with divergent cervical sperm transport: a focus on metabolites involved in amino acid metabolism

Abstract

Introduction: Cervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used.

Objectives and methods: This study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility).

Results: A total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset (P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively (P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS (P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds (P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle.

Conclusion: The suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

Keywords: Cervix; Metabolome; Microbiota; Ovine.

Fig. 1

Experimental model showing the oestrous synchronisation design and sheep cervical mucus collection. Cervical mucus was collected from Suffolk, Belclare, Fur and Norwegian White Sheep ewes (n = 28 to 30 ewes per breed) at the follicular phase of a synchronised (14 day progestogen vaginal sponge + 400 IU equine chorionic gonadotropin at sponge removal) which was followed by a natural cycle (ewes were checked for signs of oestrous using teaser rams with aprons fitted twice per day during 6 days). Each cycle was repeated 3 times (using the same ewes) over a period of 6 months. After each mucus collection, samples were stored at 20 °C. Prior to the metabolomic analysis, samples were thawed on ice and pooled within breed and with type of the cycle (4–5 ewes over 3 replicates)

Fig. 2

Pie chart showing the percentage of metabolites in each super pathway in sheep cervical mucus from all the four ewe breeds (Suffolk, Belclare, Fur and Norwegian White Sheep) at the follicular phase of both types of oestrous cycle

Fig. 3

Principal component analysis (PCA) of total metabolites analysed in four ewe breeds (indicated with different colours) at both a natural and a synchronised cycle, represented as circles and triangles, respectively. Each dot represents a pooled sample. Significant separation based on the ewe breed suggesting that there were a number of significantly different metabolites between breeds that had high and low pregnancy rates. NWS = Norwegian White Sheep

Fig. 4

Box plots of N-methylhydantoin, N-carbamosylsarcosine, 3-indoxyl sulfate and 2-oxoarginine which all had a significant ewe breed by type of the cycle interaction. The central horizontal line represents the median value with outer boundaries depicting the upper and lower quartile limits. Error bars depict the minimum and maximum distributions, with + representing the mean value and ○ the extreme data point. Fold change differences are based on comparison of median values. Significant differences (P < 0.05) between the reference level (Suffolk) and the other ewe breeds at the natural cycle are denoted with different lower case superscripts (ab) while significant differences (P < 0.05) between them at the synchronised cycle are denoted with different upper case superscripts (AB). Abbreviations: Nat = natural cycle; Syn = synchronised cycle. NWS = Norwegian White Sheep

Fig. 5

Box plots of N6-acetyllysine, cystine, cysteine s-sulfate and methionine sulfoxide which were all significantly affected by ewe breed. The central horizontal line represents the median value with outer boundaries depicting the upper and lower quartile limits. Error bars depict the minimum and maximum distributions, with + representing the mean value and ○ the extreme data point. Fold change differences are based on comparison of median values. Significant differences (P < 0.05) between the reference level (Suffolk) and the other ewe breeds at the natural cycle are denoted with different lower case superscripts (ab) while significant differences (P < 0.05) between them at the synchronised cycle are denoted with different upper case superscripts (AB). Abbreviations: Nat = natural cycle; Syn = synchronised cycle. NWS = Norwegian White Sheep

Fig. 6

Box plots of the amino acids cadaverine, putrescine, N-acetylmethionine sulfoxide and lanthionine which were all significantly different between a natural and a synchronised oestrus. The central horizontal line represents the median value with outer boundaries depicting the upper and lower quartile limits. Error bars depict the minimum and maximum distributions, with + representing the mean value and ○ the extreme data point. Fold change differences are based on comparison of median values. ^abDifferent superscripts differ significantly between the natural and synchronised cycle within ewe breed (P < 0.05). Abbreviations: Nat = natural cycle; Syn = synchronised cycle. NWS = Norwegian White Sheep