A method for promoting follicular development in cattle by injection of long acting recombinant follicle stimulating hormone into the mesoderm of the vulva

Abstract

The present application belongs to the technical field of cattle breeding, and provides a method for promoting the development of bovine follicles by injecting long-acting recombinant follicle-stimulating hormone into the urogenital mesoderm. The method comprises injecting an effective dose of long-acting recombinant follicle-stimulating hormone into the urogenital mesoderm of a cow. By injecting long-acting recombinant follicle-stimulating hormone (LArF) into the urogenital mesoderm, slow, continuous and direct action can be achieved, while off-target effects and pain are minimized. In addition, by using the minimum injection volume, the risk associated with the injection operation can be further reduced. In addition, the urogenital mesoderm injection scheme has the advantages of simpler operation, higher safety, less stress, more efficient process and lower cost under the premise of ensuring the follicle size and oocyte number required for oocyte collection.

Description

Technical Field

[0001] This invention belongs to the field of bovine breeding technology, specifically, it relates to a method for promoting bovine follicle development by injecting long-acting recombinant follicle-stimulating hormone into the vulva mesoderm. Background Technology

[0002] Oocytes are female reproductive cells that provide half of the genetic material for offspring, playing a crucial role in population reproduction. [1] The contribution of superior female livestock to the genetic imprinting of the population depends on the number of eggs ovulated, the fertilization rate of oocytes, and the development rate. [2] In nature, there are both single-ovulatory and multi-ovulatory species, and the number of offspring they produce varies. [3] However, major livestock species such as cattle are single-ovulating species. [4] Therefore, due to the physiological limitation of single ovulation, the number of calves that genetically superior cows can reproduce naturally throughout their lives is limited. [5] By regulating ovarian function through assisted reproductive technology, multiple follicles can be developed simultaneously. [6] Furthermore, oocyte retrieval unit (OPU) technology can now obtain more oocytes from follicles of different sizes (small, medium, and large). [7] Although oocyte quality varies, studies have shown that transplantable embryos can eventually be obtained through careful processing and culture during in vitro maturation. These techniques allow genetically superior cows to produce multiple embryos, thereby accelerating the realization of their breeding value.

[0003] Currently, the follicle-stimulating hormone (FSH) stimulation protocol before oocyte collection-in vitro embryo production (OPU-IVEP) is widely used, especially in Europe (88%) and North America (69.2%), which may be related to the fact that these regions are dominated by Bos taurus cattle. [8] Traditional FSH preparations are derived from animal pituitary extracts (pFSH). [9, 10] As a short-acting hormone, pFSH has a circulating half-life of only 5 hours. [11-13] Therefore, multiple injections are needed during the specific window period when the follicular wave appears in order to maintain the threshold concentration and support the growth of multiple follicles.

[14] The survival of cavitary follicles depends on the concentration and frequency of pFSH injections.

[15] Furthermore, higher doses of pFSH can improve oocyte recovery rate and embryo yield.

[16] Due to its short half-life, the traditional method requires eight injections into the donor cow.

[17] Frequent handling can cause stress in animals and poses potential risks.

[18] It may also increase the risk of technical errors and affect the overall success rate.

[19] Traditional pFSH formulations require multiple large-volume injections, and the FSH:LH ratio varies among different formulations.

[20] Besides causing tissue pain and irritation. [21, 22] Large-volume diluents (10 ml) also lead to waste of hormones, water, and adjuvants, which can be reduced through optimized solutions.

[23] Frequent animal handling also increases labor and technical support costs. Reducing handling can improve animal welfare and increase productivity.

[24] Therefore, simplifying the entire process is crucial for improving convenience, feasibility, and application effectiveness.

[0004] With the development of bioengineering technology, recombinant follicle-stimulating hormone (rFSH) has been applied in the field of human and animal reproduction.

[25] Its long half-life helps maintain a threshold concentration in the blood, preventing follicular atresia.

[26] Unlike pFSH, which has inconsistent residual luteinizing hormone (LH) levels, rFSH has high purity and can prevent premature luteinization of follicles. [27, 28] A single injection of long-acting recombinant follicle-stimulating hormone (LArF) can stimulate the development of multiple follicles in cows.

[29] Studies have shown that human rFSH can also achieve ideal reproductive results in cattle. [30-32] The novel human long-acting recombinant follicle-stimulating hormone (XOO2), independently developed in China, has a long half-life in vivo.

[33] .

[0005] Besides the hormone itself, the route of administration is also a key factor affecting drug delivery, with different routes exhibiting different distribution characteristics. Common routes of administration for FSH include intramuscular and subcutaneous injection, and in some cases, intravenous and spinal injection are also used. [34, 35] In these routes of administration, FSH must first enter the systemic circulation before reaching the target organ (i.e., the ovary).

[36] Studies have shown that FSH may also have other effects on adipose tissue, liver, bones, and other organs such as the pancreas. [37, 38] Excessive FSH may affect the physiological function of these organs through off-target effects. Vaginal administration, as an alternative delivery system, has been recognized in various reproductive applications in humans and livestock. [39-41] In humans, vaginal FSH injections have shown good results, improving patient comfort and reducing the number of visits.

[42] Because the vagina, uterus, and ovaries have specialized blood supply, vaginal administration allows drugs to preferentially act on the reproductive system. [39, 43] .

[0006] Mesotherapy is a special injection technique characterized by the slow and sustained release of drugs. Drugs injected intradermally (between the subcutaneous tissue and the dermis) are cleared from the systemic circulation more slowly than those injected deeply. This technique is used to achieve sustained drug release and is less painful compared to other routes. [36, 44, 45] . Summary of the Invention

[0007] This invention aims to reduce the number of injections and animal manipulations by using XOO2 to administer long-acting recombinant follicle-stimulating hormone (LArF) via vulvar mesotherapy (VM) in cattle. This invention provides a method for promoting bovine follicle development through vulvar mesotherapy with long-acting recombinant follicle-stimulating hormone.

[0008] To achieve the objective of this invention, this invention provides a method for promoting bovine follicle development by injecting long-acting recombinant follicle-stimulating hormone into the vulva mesoderm, the method comprising injecting an effective dose of long-acting recombinant follicle-stimulating hormone into the vulva of a cow.

[0009] Furthermore, the dosage injected per cow is 100 µg-200 µg, preferably 100 µg, 150 µg or 200 µg, more preferably 150 µg.

[0010] Further, an effective dose of long-acting recombinant follicle-stimulating hormone was dissolved in 1.5 mL of physiological saline, and the resulting injection solution was injected into the vulvar mesoderm of the bovine vagina in two separate injections at the 3 o'clock and 9 o'clock positions of the vulva.

[0011] Preferably, the injection is made into the vaginal mesodermal layer at a 30° angle, with an injection depth of 1-2 mm.

[0012] Furthermore, the injection is performed after the cattle have undergone estrus synchronization treatment, when new follicular waves appear.

[0013] Furthermore, the method includes the following steps: (1) The date on which CIDR is implanted in cattle is recorded as t. On day t+7, the progesterone plug is removed and each cow is injected with 25 mg PGF2α. For example, 25 mg of PGF2α is dissolved in physiological saline to prepare 20 mL of injection solution and injected into the cow.

[0014] (2) On day t+19, each cow was implanted with CIDR and injected with 20 μg of gonadotropin-releasing hormone (GnRH); for example, 20 μg of GnRH was dissolved in physiological saline to make 20 mL of injection solution and injected into the cow.

[0015] (3) On day t+20, each cow was given an effective dose of long-acting recombinant follicle-stimulating hormone via vulvar mesotherapy.

[0016] The long-acting recombinant follicle-stimulating hormone used in this invention is a human long-acting recombinant follicle-stimulating hormone (such as XOO2).

[0017] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention delivers long-acting recombinant follicle-stimulating hormone (LArF) via vulvar mesotherapy, achieving a slow, continuous, and direct effect while minimizing off-target effects and pain. Furthermore, this method further reduces the risks associated with the injection procedure by using the smallest possible injection volume.

[0018] Vulvar mesotherapy injection achieves targeted drug delivery to the reproductive system through the "first-pass effect." This invention uses vulvar mesotherapy injection (LArF-VM) of human long-acting recombinant follicle-stimulating hormone (FSH) in synchronized pre-treatment cows for ovarian stimulation, and compares this single-dose regimen with the traditional intramuscular multiple-injection regimen of short-acting pituitary extract FSH (SApF-IM). In the dosage optimization experiment (n=20 Holstein cows), the 150 μg dose of long-acting recombinant FSH vulvar injection regimen significantly increased the total number of follicles (18.6±3.84), the number of medium-sized follicles (12.8±2.78), and the number of oocytes collected (15.6±2.7) compared to the 100 μg, 200 μg dose groups and the traditional SApF-IM regimen group (total dose 500 μg, divided into 8 injections). P The LArF-VM protocol (≤0.05) maintained a more favorable follicle distribution: small follicles (0-3mm) accounted for 21.8%, medium follicles (3-8mm) accounted for 69.57%, and large follicles (>8mm) accounted for 8.70%; while in the SApF-IM group, medium follicles accounted for 49.23% and large follicles accounted for as much as 40%. Subsequent validation experiments (n=13) also showed that the novel optimized LArF-VM protocol and the SApF-IM protocol did not differ significantly in follicle response (22.17±7.9 vs 21.71±9.4) and the number of oocytes retrieved (15.67±6.022 vs 15.86±7.151).

[0019] This invention reduces the number of animal procedures from 12 to 5, and shortens the total injection time from 72 minutes to 30 minutes. P ≤0.01), the injection diluent volume was reduced from 10 mL to 1.5 mL, and the labor cost per donor cow was significantly reduced. P ≤0.01). The single-dose vulvar mesotherapy protocol offers advantages such as simpler operation, higher safety, less stress, more efficient process, and lower cost, while ensuring the required follicle size and number of oocytes for oocyte retrieval. Attached Figure Description

[0020] Figure 1This is a preferred embodiment of the invention showing the cow's vulva structure and the relative position of the LArF-VM injection site after alcohol disinfection.

[0021] Figure 2 This is a CIDR pre-synchronization process in a preferred embodiment of the present invention.

[0022] Figure 3 The total number of follicles and oocyte recovery in different LArF-VM dosage groups and SApF groups are shown in the preferred embodiment of the present invention.

[0023] Figure 4 The present invention provides a preferred embodiment of the total number of follicles and their population distribution in different groups based on follicle size (diameter).

[0024] Figure 5 This invention provides a comparison of the operational efficiency and labor costs of the novel LArF-VM scheme and the SApF scheme in a preferred embodiment of the present invention.

[0025] Figure 6 The ultrasound images shown in the preferred embodiment of the present invention are ovarian follicles of dairy cows in the (A) LArF-VM group and (B) SApF-IM group. Detailed Implementation

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] The human long-acting recombinant follicle-stimulating hormone (XOO2) used in the following examples was purchased from Beijing ImmunoArk Pharmaceutical Technology Co., Ltd. (Beijing, China), and the short-acting pituitary follicle-stimulating hormone (SApF) was purchased from Stimufol®, Reprobiol (Belgium).

[0028] PGF2α (prostaglandin F2α) and GnRH (gonadotropin-releasing hormone) were purchased from Ningbo 3SBio Co., Ltd.

[0029] Example This embodiment provides a low-intensity protocol involving vulvar mesotherapy with long-acting recombinant follicle-stimulating hormone (such as XOO2), and compares it with the SApF-IM protocol to explore its application effect in ovarian stimulation and oocyte collection in dairy cows.

[0030] 1. Materials and Methods This invention has been rigorously reviewed and approved by the Research Ethics Committee of the Chinese Academy of Agricultural Sciences (Beijing, China) (Approval No.: 2024 / 08 / 21).

[0031] 1.1 Experimental Animals The dairy cows used in this invention were sourced from a local ranch in the Xinjiang Uygur Autonomous Region of China. A total of 33 Holstein cows, aged 2-4 years, with body condition scores ranging from 2.5 to 3.25, were selected. Rectal and ultrasound examinations confirmed that all cows had normal reproductive function and healthy reproductive tracts. The experimental animals were fed a balanced diet twice daily (containing corn silage, alfalfa hay, wheat straw, concentrate, and vitamin-mineral premix), and had free access to clean drinking water.

[0032] 1.2 Experimental Design and Treatment Scheme This invention comprises two main experiments: (1) optimization of novel LArF(XOO2) ovarian stimulation dosage based on LArF-VM ( Figure 1 (2) Verify the ovarian response effect of the optimal stimulation dose of LArF and compare it with the traditional short-acting pituitary follicle-stimulating hormone (SApF) multiple injection regimen.

[0033] All experimental animals were initially pre-synchronized using a 7-day CIDR progesterone plug synchronization protocol. The progesterone plug was removed on day 7 after CIDR implantation, and each animal was injected with 25 mg of PGF2α solution (25 mg of PGF2α dissolved in physiological saline to prepare 20 mL of injection solution, which was then administered to the cattle). In the pre-synchronization protocol, day 9 was designated as the onset of estrus. Ten days later (day 19), all animals were considered to have exhibited new follicular waves (…). Figure 2 ).

[0034] In the first trial (dosage optimization), 20 pre-synchronized dairy cows were randomly divided into four groups based on the type and dosage of FSH they received: Group 1 was the LArF-VM (100 μg) group, Group 2 was the LArF-VM (150 μg) group, and Group 3 was the LArF-VM (200 μg) group, receiving injections of 100 μg, 150 μg, and 200 μg of LArF (XOO2), respectively. The first three groups were diluted with 1.5 mL of physiological saline and evenly divided into two VM injections. For example, in the 100 μg dose group, 50 μg of the drug was injected into the left labia and 50 μg into the right labia to ensure uniform drug distribution and optimal absorption.

[0035] The control group (Group 4) received conventional superovulation treatment using short-acting pituitary FSH (SApF) at a total dose of 500 μg. The pituitary follicle-stimulating hormone (pFSH, trade name Stimufol, Reprobiol, Belgium) was supplied in 500 μg doses, with 10 mL of diluent (physiological saline), administered intramuscularly in 8 divided doses. Figure 2Before the vulvar mesotherapy injection, the treatment group reconstituted the lyophilized XOO2 long-acting FSH with physiological saline to the required concentration.

[0036] On day 19 (day 10 of the subsequent estrus cycle), each animal was implanted with a CIDR progesterone suppository and injected with 20 μg of gonadotropin-releasing hormone (GnRH) (20 μg of GnRH was dissolved in physiological saline to prepare 20 mL of injection solution, which was then injected into the cattle). On day 20, after disinfection with 75% alcohol, each group was injected with the corresponding dose of LArF (100 μg, 150 μg, and 200 μg) via VM. Figure 1 , Figure 2 Each dose of LArF is administered in two injections: using a 30-gauge × 1 / 2-inch needle and a 1 mL syringe, inject at a 30° angle into the vulvar mesoderm to a depth of 1–2 mm, at the 3 o'clock and 9 o'clock positions on the vulva. Figure 1 , Figure 2 ) [46, 47] .

[0037] This invention preferentially selects an injection site that ensures sustained release of long-acting FSH and promotes the first-pass effect of the drug on the reproductive system. The vulva region possesses mesodermal tissue structure (facilitating drug retention) and a rich vascular distribution (promoting efficient drug absorption and transport to the reproductive organs), therefore the vulvar mesoderm was chosen as the injection site. Mesodermal tissue is a standard site for drug delivery in interstitial therapy, a characteristic that makes it suitable for sustained-release formulations. Therefore, this invention compares vulvar mesodermal injection with conventional intramuscular injection to evaluate its application effect in bovine ovarian stimulation. The results show that the innovative ovarian stimulation method provided by this invention can elicit similar response effects while minimizing the number of injections and animal treatment volume.

[0038] The control group animals also received a CIDR progesterone suppository and an intramuscular injection of 20 μg GnRH on day 19. On days 20, 21, 22, and 23, the control group received eight intramuscular injections of SApF at a gradient dose, administered twice daily. The specific method is as follows: In the control group, animals were first placed with a CIDR progesterone suppository and then injected intramuscularly with 20 μg of GnRH. Superovulation was induced using short-acting pituitary FSH (SApF, trade name Stimufol, i.e., pFSH). 500 μg of the drug was reconstituted with 10 mL of diluent and administered intramuscularly in 8 divided doses over 4 days (days 20-23). ​​The 8 injections followed a decreasing dosing regimen, administered twice daily, as follows: Day 1: 1.6 mL × 2 times Day 2: 1.4 mL × 2 times Day 3: 1.0 mL × 2 times Day 4: 1.0 mL × 2 times On day 24, all animals in both the treatment and control groups underwent ultrasound examination prior to oocyte collection to assess ovarian follicle development and size. Oocytes were recovered using OPU and counted under a stereomicroscope. The optimal dose of long-acting recombinant follicle-stimulating hormone (LArF) was determined by comparing follicle development and oocyte recovery volume with the control group.

[0039] Finally, all animals in both the treatment and control groups underwent ultrasound examination and oocyte collection on day 24. Figure 2 Before the puncture, ovarian follicle development was observed and follicle size was recorded. The number of oocytes collected via OPU was counted under a stereomicroscope. The optimal dose of LArF was determined by comparing follicle development and oocyte recovery rate with the control group.

[0040] 1.3 Follicular development and oocyte recovery Follicle counting and oocyte collection were performed simultaneously using a real-time ultrasound diagnostic instrument equipped with a 4.0–9.0 MHz micro-convex array probe, along with a catheter and a negative pressure collection device (bovine OPU negative pressure collection pump, 115 volts). Immediately before oocyte collection, the ovaries were scanned with ultrasound, and the diameter of all visible follicles was measured using the instrument's built-in scale. Follicles were then classified according to diameter into small follicles (0–3 mm), medium follicles (3–8 mm), and large follicles (>8 mm). A 20-gauge needle connected to a catheter, along with a dedicated micro-convex array vaginal probe, was used to attempt aspiration of all visible follicles. Oocyte aspiration in the two experiments was performed by different operators.

[0041] 1.4 Oocyte recovery rate Oocytes collected via follicular puncture were examined under a stereomicroscope, and relevant data were recorded and the oocyte collection rate of each group was calculated.

[0042] 1.5 Animal Handling and Economic Analysis This invention calculates parameters including the total number of animal operations throughout the entire process and compares relevant parameters between the novel and traditional schemes. The labor costs for both schemes are estimated based on the average wage standards of relevant positions for Chinese technical experts and animal handlers. The diluent dosage per donor cow in both schemes is also compared.

[0043] 1.6 Optimal Dosage Validation In the validation experiment, the single optimal dose of LArF was screened from the dosage optimization experiment. The same pre-synchronization protocol was repeated on two groups of dairy cows to further validate the effect of this LArF dose on the multiple SApF injection protocol. One group (n=6) received the optimal dose of LArF (XOO2), while the other group (n=7) served as a control group, treated with the traditional SApF protocol. All visible follicles were counted and aspirated using oocyte collection techniques to recover oocytes, which were then counted under a stereomicroscope.

[0044] 1.7 Data Analysis Data were processed and statistically analyzed for comparison. Software was used to statistically compare and plot the total number of follicles and oocyte collection rate. Normally distributed data are expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparisons between groups, followed by Tukey's test. The follicle population was classified, and the proportion of each type was calculated. Data related to operational parameters, labor costs, diluent volume, and dosage optimization experiments were compared between groups using t-tests. The statistical significance level was set at 100% (significantly significant). P ≤0.05) and highly significant ( P ≤0.01).

[0045] 2. Results In the dosage optimization experiment, the optimal dose of LArF (XOO2) injected via the vulva was determined and compared with the traditional SApF multiple injection protocol. The results showed that the number of small follicles in all LArF dose groups was significantly higher than that in the traditional SApF-IM group (P≤0.05). The average number of small follicles in each group was as follows: LArF-VM (100 μg) group 3.4±0.55, 150 μg group 4.0±1.41, 200 μg group 3.2±1.1, while the SApF-IM (500 μg) group only had 1.40±0.55 (Table 1). Figure 3 ).

[0046] The mean number of follicles in the LArF-VM (150 μg) group was 12.80±2.78, significantly higher than that in the 100 μg group (7.2±1.48) and the SApF-IM (500 μg) control group (6.40±2.07) (P≤0.05). However, there was no significant difference in the mean number of intermediate follicles between the 150 μg and 200 μg groups; furthermore, there was no significant difference in the mean number of intermediate follicles between the 100 μg, 200 μg, and traditional SApF-IM control groups (Table 1). Figure 3 ).

[0047] There was no significant difference in the number of large follicles (>8 mm) among the three LArF dosage groups (100 μg, 150 μg, and 200 μg). Similarly, there was no significant difference in the number of large follicles between the LArF-VM 200 μg group and the SApF-IM (500 μg) group (2.6 ± 1.52 vs 5.2 ± 2.86). However, the mean number of large follicles in the SApF-IM group (5.2 ± 2.86) was significantly higher than that in the LArF (100 μg group) (1.4 ± 0.54) and 150 μg group (1.6 ± 0.89) (P ≤ 0.05) (Table 1). Figure 3 ).

[0048] In summary, the total mean number of follicles in the LArF-VM (100 μg) group, 150 μg group, 200 μg group, and SAPF-IM group were 12.0±1.41, 18.6±3.84, 15.60±4.40, and 13.00±1.41, respectively. However, the total number of follicles in the LArF-VM (150 μg) group was significantly higher than that in the LArF-VM (100 μg) group and the SAPF-IM (500 μg) group (P≤0.05). However, there was no significant difference between the LArF-VM (150 μg) group and the 200 μg group; furthermore, there was no statistically significant difference in the total number of follicles among the 100 μg group, the 200 μg group, and the SAPF-IM (500 μg) group (Table 1). Figure 3 ).

[0049] The LArF-VM (150 μg) group showed better follicle recruitment, with a total of 92 follicles recruited, of which small follicles accounted for 21.74%, medium follicles for 69.57%, and large follicles for 8.70%. The low-dose LArF-VM (100 μg) group recruited 60 follicles, with the following proportions: small follicles 28.33%, medium follicles 60%, and large follicles 11.67%. The high-dose LArF-VM (200 μg) group recruited 72 follicles, with small follicles 20.78%, medium follicles 62.34%, and large follicles 16.88%. Meanwhile, SApF-IM (500 μg) only recruited 65 follicles, with the following proportions: small follicles 10.77%, medium follicles 49.23%, and large follicles 40% (Table 1). Figure 4 ).

[0050] Table 1. Total number of ovarian follicles and oocyte recovery in dairy cows under different LArF-VM dosage treatments (compared with the SApF control group) Note: Different letters following the data in the same row indicate significant differences. P <0.05, where the letters are the same, indicates that the difference is not significant ( P >0.05). The same applies below.

[0051] Based on established classification criteria, this invention grouped the follicle population according to follicle size and compared the mean follicle diameter of different doses of LArF-VM (100 μg, 150 μg, 200 μg) with the 500 μg SApF-IM control group. The small follicle diameter data are as follows: LArF-VM 100 μg group: 2.55±0.38 mm; 150 μg group: 2.25±0.51 mm; 200 μg group: 2.51±0.49 mm; SApF-IM 500 μg group: 1.90±0.57 mm. There was no significant difference in the mean small follicle diameter among the three LArF-VM groups; however, the mean small follicle diameter in the SApF-IM 500 μg group was significantly smaller than that in the 100 μg and 200 μg groups (P≤0.05), but not significantly different from the 150 μg group. For mid-sized follicles, the mean diameter of the conventional SApF-IM control group (5.43±1.60 mm) was significantly larger than that of all LArF-VM dosage groups (100 μg, 150 μg, 200 μg) (P≤0.05), while there was no significant difference in the mean diameter of mid-sized follicles among the three LArF-VM dosage groups. The mean diameters of large follicles were as follows: LArF-VM 100 μg group: 11.00±1.87 mm; 150 μg group: 8.87±1.38 mm; 200 μg group: 10.40±1.70 mm; SApF-IM 500 μg group: 9.54±1.01 mm. There was no significant difference in the mean diameter of large follicles between any of the LArF dosage groups and the control group (Table 2).

[0052] Table 2. Follicle diameters of different follicle types in the LArF-VM treatment group and the control group.

[0053] Consistent with the total number of follicles, the number of oocytes recovered also showed a significant trend among the groups. The number of oocytes recovered in the LArF-VM 150 μg group (15.60 ± 2.7) was significantly higher than that in other LArF-VM dosage groups, and significantly higher than that in the SApF-IM 500 μg control group. P(≤0.05). There was no significant difference in the number of oocytes recovered among the LArF-VM 100 μg group, 200 μg group, and SApF-IM 500 μg control group, with values ​​of 10.0±2.45, 11.00±1.87, and 10.80±1.92 respectively (Table 1). In summary, the LArF-VM 150 μg group had the highest total number of follicles and the highest number of oocytes recovered. Therefore, in the second experiment, LArF-VM 150 μg was determined as the optimal dose for further validation and comparison with the traditional SApF-IM ovarian stimulation protocol.

[0054] This invention compares the novel single-injection LArF-VM protocol with the traditional multiple-injection SApF-IM protocol. Results showed that the LArF-VM protocol significantly reduced animal treatment frequency, shortened work time, and reduced the number of injections from 8 to 1. All groups underwent pre-synchronization treatment followed by ovarian stimulation: the total number of treatments in the LArF-VM group was significantly reduced to 5, while in the SApF group it was 12. P ≤0.01); The injection time per cow in the LArF-VM group was significantly shorter, significantly lower than the traditional multi-injection SApF-IM regimen (30 minutes vs 72 minutes). P ≤0.01). Referring to the average daily wage of 940 yuan for skilled workers and 340 yuan for ordinary laborers in China, the calculation shows that the total cost of the new LArF-VM scheme for each group of 5 dairy cows is significantly reduced to only US$179.94 (approximately 1280 yuan), while the traditional SApF-IM scheme costs US$287.91 (approximately 2048 yuan). P ≤0.01)( Figure 4 Furthermore, the diluent usage of the novel single-injection regimen (1.5 mL per cow) is significantly lower than that of the traditional multi-injection SApF-IM regimen (10 mL per cow). P ≤0.01)(Tables 3-4, Figure 5 ). Figure 6 Ultrasound images show ovarian follicles in cows of group (A) LArF-VM and group (B) SApF-IM.

[0055] Table 3 Comparison of resource utilization parameters between the novel LArF-VM scheme and the SApF-IM scheme of this invention.

[0056] Table 4. Comparison of follicular response and oocyte retrieval in validation experiments between the novel LArF-VM protocol of this invention and the traditional SApF-IM protocol.

[0057] Two experimental results demonstrate that the novel LArF-VM protocol achieves equivalent efficacy to the traditional protocol in bovine ovarian stimulation. Long-acting recombinant follicle-stimulating hormone (LArF) maintains the optimal FSH level required for ovarian stimulation with a significantly reduced number of injections. Furthermore, the vaginal vestibule (VM) administration route allows for direct action on the reproductive system through the first-pass effect, providing an ideal injection site for targeted drug delivery. [44, 45] Previously, there were no reports on the application of LArF in cattle via the VM pathway. This invention aims to establish a simplified, low-stress, and efficient protocol for the OPU-IVEP process in dairy cows. Reducing the number of animal treatments not only lowers operational stress but also optimizes the rationality of overall human resource allocation. The results show that the LArF-VM protocol can be effectively applied to ovarian stimulation in dairy cows with minimal animal treatments. All LArF-VM dose groups showed a significantly higher number of small antral follicles (0–3 mm), possibly due to the slow and continuous release of FSH achieved through the VM pathway, maintaining a stable follicle recruitment threshold. There were no significant differences among the three LArF-VM dose groups, while the SApF-IM group consistently showed a lower number of small follicles. This phenomenon may be related to the fluctuations in FSH levels caused by frequent injections: fluctuating hormone levels promote small follicle atresia. Numerous studies have shown that after the emergence of a dominant follicle, secondary small follicles are subject to growth inhibition and apoptosis. [48-52] Studies suggest that repeated injections of the traditional SApF protocol lead to high FSH spikes, triggering small follicle atresia. Notably, the high-dose LArF-VM (200 μg) group maintained a large number of small follicles while also developing more large follicles, indicating that both long-acting recombinant FSH itself and the VM administration route are more efficient at maintaining a small follicle pool than the traditional SApF protocol. However, the underlying mechanisms of this phenomenon require further investigation. Although some reports indicate that oocytes derived from small follicles have lower developmental potential...

[53] However, an increase in the number of small follicles still helps to improve the total number of oocytes recovered per donor cow—studies have shown that these small follicles can be subsequently aspirated and recovered 3-4 days after the first OPU. [54-56] Medium-sized follicles (3-8 mm in diameter) are considered the optimal follicle size category for OPU-IVEP technology.

[48] Cumulus-oocyte complexes (COCs) produced by follicles of this size have superior developmental potential during the IVEP process.

[53] Experimental data from this invention show that the proportion of medium-sized follicles in the 150 μg LArF-VM group was significantly higher than that in the 100 μg dose group and the traditional SApF (500 μg) group, but there was no significant difference compared with the 200 μg dose group, which is consistent with the typical inverted U-shaped dose-response relationship of LArF. These results indicate that 150 μg LArF administered via the VM route provides sufficient and continuous recombinant FSH exposure, effectively recruiting small antral follicles and promoting their development to the ideal range of 3–8 mm, while avoiding the dominance or atresia of co-developing follicles that may result from overstimulation. Consistent with previous studies, a moderate FSH dose is more beneficial in promoting follicle development to the optimal diameter range required for OPU.

[48] Therefore, the 150 μg LArF-VM regimen is more suitable for inducing mid-stage follicle development compared to other dosage groups and traditional regimens. Excessive doses may not only be wasteful but also have adverse effects. In summary, administration of 150 μg LArF (XOO2) via the VM route can effectively increase the number of mid-stage follicles recovered. The number of large follicles (>8 mm) in the traditional SApF-IM group was significantly higher than that in the low-dose and medium-dose LArF-VM groups (100 μg and 150 μg), but there was no significant difference compared to the high-dose LArF-VM (200 μg) group. This result reflects that high-dose FSH can accelerate follicle development to the large follicle stage.

[48] Furthermore, the pFSH product used in the SApF group contained 20% residual pituitary luteinizing hormone (pLH).

[57] FSH initiates early follicular development, while LH begins to play a role when the follicle diameter reaches 8 mm.

[58] The follicle is primarily responsible for the further maturation of follicles. Studies have shown that compared with pFSH treatment alone, pFSH combined with LH treatment can significantly increase the number of follicles with a diameter greater than 10 mm.

[59] Therefore, the study speculates that the presence of pLH in the traditional protocol may be one of the reasons for the higher number of large follicles in the SApF group. However, high FSH levels may also lead to premature luteinization of large follicles, causing follicular dysfunction and reducing reproductive efficiency.

[60] .

[0058] Although there was no significant difference in total follicle size among the three LArF-VM dose groups, the diameter of the intermediate follicle in the SApF-IM group was significantly larger than that in all LArF-VM dose groups. P≤0.05). This phenomenon reflects the accelerating effect of high-dose pulsatile FSH exposure on follicle growth in dairy cows, rather than the stable low-amplitude hormone release effect achieved through the VM pathway. Furthermore, residual LH (luteinizing hormone) in pFSH preparations may also be one of the reasons for the larger follicle diameter. [57, 59] .

[0059] Overall, the average total number of follicles in the LArF-VM (150 μg) group was significantly higher than that in the SApF group, with a higher proportion of small and medium-sized follicles. This further supports the suitability of 150 μg LArF administration via the VM route for ovarian stimulation in dairy cows. This effect may stem from the continuous and stable FSH supply achieved through vaginal vestibular administration, and the fact that rFSH is more effective than pituitary FSH (pFSH) in recruiting small follicles (non-ovulatory follicles).

[61] The results of different doses of LArF in this invention are consistent with previous studies on rFSH. [62, 63] More importantly, this response may be related to the rFSH-specific ovarian superovulation effect, the specific mechanism of which still needs further elucidation. On the other hand, the presence of a dominant follicle may inhibit the growth of co-developing follicles and lead to their degeneration. [64-66] Therefore, the study speculates that the smaller number of large follicles in the LArF-VM (150 μg) group may be one of the reasons for its higher antral follicle recruitment efficiency.

[0060] The trends in oocyte recovery rates across groups were largely consistent with the follicular response: the LArF-VM 150 μg group recovered significantly more oocytes during OPU than the traditional SApF protocol and other LArF dosage groups (100 μg and 200 μg). This result is clearly due to the higher number of small and medium-sized follicles in this group—multiple studies have confirmed that small and medium-sized follicles can yield more high-quality oocytes through OPU.

[67] Another possible reason is that the group has a smaller number of dominant follicles.

[0061] Studies have shown that OPU performed in the absence of dominant follicles significantly improves both oocyte recovery rate and quality.

[68] Although limited by experimental conditions, this invention was unable to assess oocyte quality using IVEP. However, existing literature indicates that oocytes derived from medium-sized follicles are suitable for IVEP, and oocytes derived from small follicles also possess significant developmental potential. Therefore, an increasing number of studies have challenged the traditional view that oocytes from small follicles have poorer quality. [69, 70] FSH stimulation during the appearance of follicular waves can increase the number of intermediate follicles and improve oocyte quality. [48,70]This invention, through pre-synchronization treatment of animals and stimulation on day 10 of the estrous cycle, showed that the optimal dose of 150 μg LArF-VM significantly increased the number of follicles and the amount of oocytes recovered. In summary, the LArF-VM (150 μg) regimen, by increasing the number of small and medium-sized follicles and reducing the formation of dominant follicles, combined with a simplified administration method in pre-synchronized dairy cows, outperforms the traditional SApF regimen in terms of oocyte production.

[0062] In the validation experiment, the optimal dose of 150 μg LArF-VM was compared with the traditional SApF-IM multiple injection protocol in another batch of dairy cows. The results showed no statistically significant difference in the total number of follicles and the number of oocytes recovered between the two groups. However, compared with the dose optimization experiment, the number of follicles and the oocyte recovery rate in the LArF-VM (150 μg) group were slightly lower, which may be related to the different XOO2 batches used in the two experiments. Nevertheless, the results of the LArF-VM group in the validation experiment were still superior to those of the SApF-IM group, and considering its significantly reduced operational requirements, the application value of this protocol remains fully justified.

[0063] In the experiment, both the control and treatment groups were injected with PGF2α and GnRH to synchronize estrus and initiate follicular activity. These hormones primarily promote the appearance of follicular activity, while follicles grow under the influence of FSH over the subsequent four days. Long-acting recombinant follicle-stimulating hormone (LArF) plays a crucial role in maintaining the number of small and medium-sized follicles by inhibiting follicular atresia. The increased number of small and medium-sized follicles indicates that FSH has sustained activity in supporting follicular growth.

[0064] The dosage of long-acting FSH was optimized to maintain the ideal follicle diameter for the optimal OPU. Results showed that, compared to the conventional intramuscular injection protocol, transvaginal mesotherapy with long-acting FSH effectively maintained follicle count and oocyte production. Furthermore, the average number of large follicles (>8 mm) produced by high-dose long-acting FSH (200 µg) was comparable to that of the conventional protocol, indicating that long-acting FSH has a clear biological effect, and that a medium dose (150 µg) of long-acting FSH is sufficient to produce follicles suitable for the OPU.

[0065] The rational use of resources is crucial for the sustainability and economic benefits of embryo production. The efficient allocation of human and material resources can significantly reduce the unit cost of embryo production. [71, 72] Compared to the traditional SApF-IM multi-injection regimen, the novel LArF-VM regimen significantly reduces the number of animal treatments, which not only reduces the risk of injury and stress levels in donor cattle.

[73] It also improves animal welfare. Operationally, reducing repetitive treatments and frequent injections saves technicians time and manpower; each animal only requires 2-6 minutes of injection time, which significantly reduces costs for large-scale group farming.

[74] Although previous studies have attempted to mix pFSH with adjuvants to prolong absorption time and achieve single-dose administration. [75-77] However, the use of adjuvants may induce the production of anti-FSH antibodies, affecting the efficacy of subsequent treatments.

[78] In contrast, the LArF-VM regimen can directly target the reproductive system, avoiding initial drug loss at metabolic sites. While ensuring equivalent efficacy, it has significant advantages such as ease of operation, lower cost, and better animal welfare, making it more promising than traditional multi-injection regimens.

[0066] The novel LArF-VM regimen requires only 1.5 mL of diluent per animal, compared to 10 mL for the traditional SApF-IM multi-injection regimen. This smaller volume of liquid injected via the VM route significantly reduces discomfort and injection pain in animals.

[79] Further development could involve creating a precision injection pen device for drug delivery to dairy cows' VM (vegetative state) systems to improve drug delivery accuracy.

[80] Reducing the amount of diluent used also helps conserve drinking water resources and lower the production and related costs of pharmaceutical-grade water.

[81] .

[0067] Despite rigorous purification and testing, pFSH products cannot be guaranteed to be free of pathogen contamination. These products are derived from thousands of slaughterhouse animals with unknown health conditions, and any source could potentially transmit pathogens. [82, 83] In contrast, rFSH is produced under strict GMP conditions through mammalian cell culture, virtually eliminating the risk of transmission of animal-derived pathogens.

[84] Developing reproduction and conservation solutions based on the safer rFSH. Replacing slaughterhouse-derived pFSH with rFSH can reduce biohazards and support sustainable, biosafe food animal production globally.

[0068] In summary, the LArF-VM ovarian stimulation protocol offers advantages such as greater safety, convenience, low stress, high efficiency, and cost-effectiveness, while achieving ideal OPU results. However, the oocyte quality of this protocol still needs further validation through blastocyst development rate, and future research should focus on embryonic developmental potential and pregnancy rate. Furthermore, it is speculated that this low-treatment protocol can be extended to wild or semi-wild bovine species such as yaks that are difficult to restrain and require minimal treatment.

[85] .

[0069] Based on the concept that vulvar mesotherapy is an effective way to achieve continuous FSH delivery in cattle, this invention has successfully developed a simplified “1, 2, 3 GO” ovarian stimulation protocol: 150 μg of long-acting recombinant FSH (XOO2) is injected via vulvar mesotherapy. This protocol can achieve the same follicle development and oocyte retrieval effects as the traditional 8-injection short-acting pituitary FSH protocol. Note: After CIDR removal, all animals will ovulate on day 9, which will be regarded as the estrus day of the subsequent estrous cycle. 10 days later (day 19, i.e., day 10 of the subsequent cycle), the animals will be divided into two groups and receive the corresponding ovarian stimulation protocol. For ease of understanding, day 19 is regarded as day 1 of ovarian stimulation. The protocol includes 3 simple steps: (1) implanting CIDR progesterone plug and injecting GnRH on day 1; (2) injecting LArF (XOO2) on day 2; (3) oocyte retrieval 3 days later to obtain oocytes (Get=G, Oocyte=O). Therefore, the scheme is named "1,2-3 GO scheme".

[0070] Furthermore, the new LArF-VM protocol significantly reduces the number of treatments, injection time, diluent usage, and labor costs per donor cow, while improving animal welfare and safety. These improvements make this new protocol a safer, less stressful, more efficient, and cost-effective option for OPU operations in bovine breeding projects.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

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Claims

1. A method for promoting bovine follicle development by injecting long-acting recombinant follicle-stimulating hormone into the vulva, characterized in that, The method involves injecting an effective dose of long-acting recombinant follicle-stimulating hormone into the mesoderm of the bovine vulva.

2. The method according to claim 1, characterized in that, The dosage injected per cow is 100 µg-200 µg, preferably 100 µg, 150 µg or 200 µg.

3. The method according to claim 2, characterized in that, The dosage injected per cow is 150 µg.

4. The method according to claim 1, characterized in that, First, dissolve an effective dose of long-acting recombinant follicle-stimulating hormone in 1.5 mL of physiological saline. The resulting injection solution is then injected twice into the vulvar mesoderm of the bovine vagina at the 3 o'clock and 9 o'clock positions.

5. The method according to claim 4, characterized in that, Inject into the vaginal mesodermal layer at a 30° angle to a depth of 1-2 mm.

6. The method according to claim 1, characterized in that, The injection was performed after the cattle underwent estrus synchronization treatment, when new follicular waves appeared.

7. The method according to claim 6, characterized in that, Includes the following steps: (1) The date on which CIDR is implanted in cattle is recorded as t. On day t+7, the progesterone plug is removed and each cow is injected with 25 mg PGF2α. (2) On day t+19, each cow was implanted with CIDR and injected with 20 μg of gonadotropin-releasing hormone; (3) On day t+20, each cow was given an effective dose of long-acting recombinant follicle-stimulating hormone via vulvar mesotherapy.

8. The method according to any one of claims 1-7, characterized in that, The long-acting recombinant follicle-stimulating hormone is a human long-acting recombinant follicle-stimulating hormone.

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